Identification and Attenuation of the Immunosuppressive Domains in Fusion Proteins of Enveloped RNA Viruses

ABSTRACT

The present invention relates to enveloped RNA viruses. The invention in particular relates to the generation of superior antigens for mounting an immune response by first identifying then mutating the immunosuppressive domains in fusion proteins of enveloped RNA viruses resulting in decreased immunosuppressive properties of viral envelope proteins from the viruses.

The present invention relates to enveloped RNA viruses. In particular,the invention relates to the generation of superior antigens formounting an immune response by first identifying then mutating theimmunosuppressive domains in fusion proteins of enveloped RNA virusesresulting in decreased immunosuppressive properties of viral envelopeproteins from said viruses.

TECHNICAL BACKGROUND Classification of Viruses ICTV Classification

The International Committee on Taxonomy of Viruses (ICTV) developed thecurrent classification system and wrote guidelines that put a greaterweight on certain virus properties to maintain family uniformity. Aunified taxonomy (a universal system for classifying viruses) has beenestablished. The 7th ICTV Report formalized for the first time theconcept of the virus species as the lowest taxon (group) in a branchinghierarchy of viral taxa. However, at present only a small part of thetotal diversity of viruses has been studied, with analyses of samplesfrom humans finding that about 20% of the virus sequences recovered havenot been seen before, and samples from the environment, such as fromseawater and ocean sediments, finding that the large majority ofsequences are completely novel.

The general taxonomic structure is as follows:

-   -   Order (-virales)    -   Family (-viridae)    -   Subfamily (-virinae)    -   Genus (-virus)    -   Species (-virus)

In the current (2008) ICTV taxonomy, five orders have been established,the Caudovirales, Herpesvirales, Mononegavirales, Nidovirales, andPicornavirales. The committee does not formally distinguish betweensubspecies, strains, and isolates. In total there are 5 orders, 82families, 11 subfamilies, 307 genera, 2,083 species and about 3,000types yet unclassified.

Baltimore Classification

The Baltimore Classification of viruses is based on the method of viralmRNA synthesis.

The ICTV classification system is used in conjunction with the Baltimoreclassification system in modern virus classification.

The Baltimore classification of viruses is based on the mechanism ofmRNA production. Viruses must generate mRNAs from their genomes toproduce proteins and replicate themselves, but different mechanisms areused to achieve this in each virus family. Viral genomes may besingle-stranded (ss) or double-stranded (ds), RNA or DNA, and may or maynot use reverse transcriptase (RT). Additionally, ssRNA viruses may beeither sense (+) or antisense (−). This classification places virusesinto seven groups:

-   -   I: dsDNA viruses (e.g. Adenoviruses, Herpesviruses, Poxviruses)    -   II: ssDNA viruses (+)sense DNA (e.g. Parvoviruses)    -   III: dsRNA viruses (e.g. Reoviruses)    -   IV: (+)ssRNA viruses (+)sense RNA (e.g. Picornaviruses,        Togaviruses)    -   V: (−)ssRNA viruses (−)sense RNA (e.g. Orthomyxoviruses,        Rhabdoviruses)    -   VI: ssRNA-RT viruses (+)sense RNA with DNA intermediate in        life-cycle (e.g. Retroviruses)    -   VII: dsDNA-RT viruses (e.g. Hepadnaviruses)

As an example of viral classification, the chicken pox virus, varicellazoster (VZV), belongs to the order Herpesvirales, family Herpesviridae,subfamily Alphaherpesvirinae, and genus Varicellovirus. VZV is in GroupI of the Baltimore Classification because it is a dsDNA virus that doesnot use reverse transcriptase.

Many viruses (e.g. influenza and many animal viruses) have viralenvelopes covering their protein cores. The envelopes typically arederived from portions of the host cell membranes (phospholipids andproteins), but include some viral glycoproteins. Functionally, viralenvelopes are used to enable viruses to enter host cells. Glycoproteinson the surface of the envelope serve to identify and bind to receptorsites on the host's membrane. Subsequently the viral envelope then fuseswith that of the host's, allowing the viral capsid and viral genome toenter and infect the host.

Typically, in RNA viruses a single transmembrane glycoprotein, a fusionprotein, undergoes a conformational transition triggered by receptorrecognition or low pH, leading to the insertion of a fusion peptide intothe plasma membrane or the membrane of an endocytic vesicle. For someRNA viruses, including members of the paramyxovirus family, separateenvelope proteins mediate attachment and fusion.

Membrane fusion can occur either at the plasma membrane or at anintracellular location following internalization of virus byreceptor-mediated endocytosis. Fusion is mediated by viral transmembraneproteins known as fusion proteins. Upon appropriate triggering, thefusion protein interacts with the target membrane through a hydrophobicfusion peptide and undergoes a conformational change that drives themembrane fusion reaction. There are a variety of fusion triggers,including various combinations of receptor binding, receptor/coreceptorbinding, and exposure to the mildly acidic pH within the endocyticpathway. Fusion proteins from different viruses have different names inspite of the common functionality.

Based on important structural features, many virus membrane fusionproteins are currently annotated to either the “class I” membrane fusionproteins exemplified by the influenza hemagglutinin (HA) or HIV-1 gp41,or the “class II” proteins of the alphaviruses and flaviviruses. Thealphaviruses and flaviviruses are members of the Togaviridae andFlaviviridae families, respectively. These small envelopedpositive-sense RNA viruses are composed of a capsid protein thatassembles with the RNA into the nucleocapsid, and a lipid bilayercontaining the viral transmembrane (TM) proteins.

Class I fusion proteins are synthesized as single chain precursors,which then assemble into trimers. The polypeptides are then cleaved byhost proteases, which is an essential step in rendering the proteinsfusion competent. This proteolytic event occurs late in the biosyntheticprocess because the fusion proteins, once cleaved are metastable andreadily activated. Once activated, the protein refolds into a highlystable conformation. The timing of this latter event is of crucialimportance in the fusion process. Maintenance of the intact precursorpolypeptide during folding and assembly of the oligomeric structure isessential if the free energy that is released during the refolding eventis to be available to overcome the inherent barriers to membrane fusion.The new amino-terminal region that is created by the cleavage eventcontains a hydrophobic sequence, which is known as the fusion peptide.The authentic carboxy-terminal region of the precursor polypeptidecontains the transmembrane anchor. In the carboxy-terminal polypeptide,there are sequences known as the heptad repeat that are predicted tohave an alpha helical structure and to form a coiled coil structure.These sequences participate in the formation of highly stable structurethat characterizes the post-fusion conformation of the fusion protein.

The class II fusion proteins are elongated finger-like molecules withthree globular domains composed almost entirely of β-sheets. Domain I isa β-barrel that contains the N-terminus and two long insertions thatconnect adjacent β-strands and together form the elongated domain II.The first of these insertions contains the highly conserved fusionpeptide loop at its tip, connecting the c and d β-strands of domain II(termed the cd loop) and containing 4 conserved disulfide bondsincluding several that are located at the base of the fusion loop. Thesecond insertion contains the ij loop at its tip, adjacent to the fusionloop, and one conserved disulfide bond at its base. A hinge region islocated between domains I and II. A short linker region connects domainIto domain III, a β-barrel with an immunoglobulin-like fold stabilizedby three conserved disulfide bonds. In the full-length molecule, domainIII is followed by a stem region that connects the protein to the virusTM anchor. Fitting of the structure of alphavirus E1 to cryo-electronmicroscopy reconstructions of the virus particle reveals that E1 islocated almost parallel to the virus membrane, and that E1-E1interactions form the an icosahedral lattice.

Immunosuppressive Properties of Enveloped Viruses with Type I FusionProteins

Fusion proteins of a subset of enveloped Type I [1] viruses (retrovirus,lentivirus and filoviruses) have previously been shown to feature animmune suppressive activity. Inactivated retroviruses are able toinhibit proliferation of immune cells upon stimulation [2-4]. Expressionof these proteins is enough to enable allogenic cells to grow to a tumorin immune competent mice. In one study, introduction of ENV expressingconstruct into MCA205 murine tumor cells, which do not proliferate upons.c. injection into an allogeneic host, or into CL8.1 murine tumor cells(which overexpress class I antigens and are rejected in a syngeneichost) resulted in tumor growth in both cases [5]. Such immunosuppressivedomains have been found in a variety of different viruses with type 1fusion mechanism such as gamma-retroviruses like Mason pfeizer monkeyvirus (MPMV) and murine leukemia virus (MLV), lentiviruses such as HIVand in filoviruses such as Ebola and Marburg viruses [6-9].

This immune suppressive activity was in all cases located to a verywell-defined structure within the class I fusion proteins, moreprecisely at the bend in the heptad repeat just N-terminale of thetransmembrane structure in the fusion protein. The immunosuppressiveeffects range from significant inhibition of lymphocyte proliferation[7,8], cytokine skewing (up regulating IL-10; down regulating TNF-α,IL-12, IFN-γ) [10] and inhibition of monocytic burst [11] to cytotoxic Tcell killing [12]. Importantly, peptides spanning ISD in these assaysmust either be linked as dimers or coupled to a carrier(i.e. >monomeric) to be active. Such peptides derived fromimmune-suppressive domains are able to reduce or abolish immuneresponses such as cytokine secretion or proliferation of T-cells uponstimulation. The protection mediated by the immunosuppressive propertiesof the fusion protein from the immune system of the host is not limitedto the fusion protein but covers all the viral envelope proteinsdisplayed at viral or cellular membranes in particular also the proteinmediating attachment of the virus to the cell.

Co-Location of the Immunosuppression Domain and the Fusion Domain

The immunosuppressive domain of retro-, lenti- and filoviruses overlap astructurally important part of the fusion subunits of the envelopeproteins. Although the primary structure (sequence) of this part of thefusion proteins can vary greatly from virus to virus, the secondarystructure, which is very well preserved among different virus families,is that of an alpha helix that bends in different ways during the fusionprocess This structure plays a crucial role during events that result infusion of viral and cellular membranes. It is evident that theimmunosuppressive domains of these (retroviral, lentiviral andfiloviral) class I fusion proteins overlap with a very important proteinstructure needed for the fusion proteins mechanistic function.

The energy needed for mediating the fusion of viral and cellularmembranes is stored in the fusion proteins, which are thus found in ameta-stable conformation on the viral surface. Once the energy isreleased to drive the fusion event, the protein will find its mostenergetically stable conformation. In this regard fusion proteins can becompared with loaded springs that are ready to be sprung. This highenergy conformation makes the viral fusion proteins very susceptible tomodifications; Small changes in the primary structure of the proteinoften result in the protein to be folded in its stable post fusionconformation. The two conformations present very different tertiarystructures of the same protein.

It has been shown in the case of simple retroviruses that smallstructural changes in the envelope protein are sufficient to remove theimmune suppressive effect without changing structure and hence theantigenic profile.

The mutated non-immune suppressive envelope proteins are much betterantigens for vaccination. The proteins can induce a 30-fold enhancementof anti-env antibody titers when used for vaccination and are muchbetter at launching an effective CTL response [6]. Furthermore, virusesthat contain the non-immunosuppressive form of the friend murineleukemia virus envelope protein, although fully infectious in irradiatedimmunocompromised mice cannot establish an infection in immunocompetentanimals. Interestingly in the latter group the non-immunosuppressiveviruses induce both a higher cellular and humeral immune response, whichfully protect the animals from subsequent challenge by wild type viruses[13].

Immunosuppressive domains in the fusion proteins (viral envelopeproteins) from Retroviruses, lentiviruses and Filoviruses have beenknown since 1985 for retrovirus [7], since 1988 for lentivirus [8] andsince 1992 for filoviruses [14]. These viruses, as mentioned above, allbelong to enveloped RNA viruses with a type I fusion mechanism. Theimmunosuppressive domains of lentivirus, retroviruses and filovirusesshow large structural similarity. Furthermore the immunosuppressivedomain of these viruses are all located at the same position in thestructure of the fusion protein, more precisely in the linker betweenthe two heptad repeat structures just N-terminal of the transmembranedomain in the fusion protein. These heptad repeat regions constitute twoalpha helices that play a critical role in the active mechanism ofmembrane fusion by these proteins. The immune suppressive domains can belocated in relation to two well conserved cystein residues that arefound in these structures. These cystein residues are between 4 and 6amino acid residues from one another and in many cases are believed toform disulfide bridges that stabilize the fusion proteins. The immunesuppressive domains in all three cases include at least some of thefirst 22 amino acids that are located N-terminal to the first cysteineresidue. Recently the immunosuppressive domains in the fusion protein ofthese viruses have been successfully altered in such a way that thefusogenic properties of the fusion protein have been preserved. Suchmutated fusion proteins with decreased immunosuppressive properties havebeen shown to be superior antigens for vaccination purposes [13].

SUMMARY OF THE INVENTION

The inventors have been able to devise methods for the identification ofnew immunosuppressive domains or potentially immunosuppressive domainslocated in proteins displayed at the surface of enveloped RNA viruses.The inventors of the present invention have surprisingly foundimmunosuppressive domains or potentially immunosuppressive domains infusion proteins in a large number of other enveloped RNA viruses inaddition to lentivirus, retrovirus and filovirus, where suchimmunosuppressive domains had not been described previously. Inaddition, the inventors have been able to develop methods for mutatingsaid immunosuppressive domains in order to reduce the immunosuppressiveproperties of viral surface proteins, which are useful for providingstrategies for producing new vaccines with improved properties by makingsuperior antigens, or for generation of neutralizing antibodies. Throughsuch approaches, the inventors have been able to propose vaccinationregimes against different types of viruses such as e.g. Hepatitis C,Dengue virus and Influenza where effective vaccination regimes have beenin great demand for many years. This may allow the production ofvaccines against virus for which no vaccines has been known e.g.hepatitis C and Dengue, as well as improved versions of known vaccines,e.g. for Influenza.

According to an aspect, the inventors propose the use of up to fourparameters for the identification of immunosuppressive domain inenveloped RNA viruses with hitherto un-described immunosuppressiveproperties. Proposed parameters used as part of a strategy foridentifying a peptide sequence or a peptide which likely acts asimmunosuppressive domains may comprise one or more of the followingparameters (preferably all parameters are used):

1): The peptide is preferably located in the fusion protein of envelopedRNA viruses;2): The peptide is preferably capable of interacting with membranes;3): Preferably a high degree of homology in the primary structure(sequence) of the peptide of said domain exists either within the Order,Family, Subfamily, Genus, or Species of viruses. This feature is due tothe immunosuppressive domain being under a dual selection pressures, oneas an immunosuppressive entity ensuring protection of the viral particlefrom the host immune system, another as a peptide interacting withmembranes;4): The position at the surface of the fusion protein at a givenconformation is preferably a feature of immunosuppressive domains. Thiscan be revealed either by position in a 3D structure or by antibodystaining of cells expressing the fusion protein or on viral surfacesdisplaying the fusion protein.

Based upon these parameters the inventors have inter alia identifiedthree new groups of enveloped RNA viruses with immunosuppressive domainsin their fusion protein:

1: The inventors have identified immunosuppressive domains amongenveloped RNA viruses with type II fusion mechanism. Hitherto,immunosuppressive domains have not been described for any enveloped RNAviruses with a type II fusion mechanism. Immunosuppressive domains havebeen identified by the inventors at two positions in two differentgroups of viruses:

-   i. Co-localizing with the fusion peptide exemplified by the    identification of an common immunosuppressive domain in the fusion    peptide of Flavivirus (Dengue virus, west Nile virus etc), and-   ii. In the hydrophobic alpha helix N-terminal of the transmembrane    domain in the fusion protein exemplified by the finding of an    immunosuppressive domain in said helixes of all flaviridae e.g.    Hepatitis C virus, Dengue, west nile etc.    2: The inventors have identified immunosuppressive domains in the    fusion protein among enveloped RNA viruses with type I fusion    mechanism (excluding lentivirus, retrovirus and filovirus). This    position co-localizes with the fusion peptide of said fusion protein    as demonstrated by the identification of a common immunosuppressive    domain in the fusion peptide of all Influenza A and B types.    3: The inventors have identified potential immunosuppressive domains    located at various positions of type I enveloped RNA viruses    (excluding lentivirus, retrovirus and filovirus) as well as in    enveloped RNA viruses featuring a fusion protein with neither a type    I nor a type II fusion structure.

After identification of the immunosuppressive domains these must bemutated in order to decrease or completely abrogate theimmunosuppressive properties of the whole envelope protein (preferablyboth the attachment and fusion part of the envelope protein if these areseparate proteins). Such viral envelope proteins with reducedimmunosuppressive properties are ideal candidates for use as antigens invaccine compositions or for the production of neutralizing antibodies.

According to an aspect, the invention concerns a method for identifyingan immunosuppressive domain of an enveloped RNA virus containing a lipidmembrane, said method comprising the following steps:

-   -   a. Identifying the fusion protein of said virus;    -   b. Identifying at least one well conserved domain preferably        among the membrane associated domains of said fusion protein        (where the immunosuppressive domain is preferably located at the        surface of the protein in one or more of the different        conformations of the fusion protein undergoing fusion);    -   c. Optionally multimerizing or dimerizing said peptide; and    -   d. Confirming the immunosuppressive activity of at least one        optionally multimerized or dimerized peptide by testing said        optionally dimerized or multimerized peptide for        immunosuppressive activity.

Concerning step a., fusion proteins or putative fusion proteins areusually identified by searching scientific databases, e.g. such assearching NCBI taxonomy database (http://www.ncbi.nlm.nih.gov/Taxonomy/)and selecting proteins of the Family, Subfamily, Genus or Species to beinvestigated and subsequently searching these for fusion, or thespecific fusion protein, such as the protein listed in Table 1 below.

Concerning step b., vira are divided according to the followingclassification: Order (-virales), Family (-viridae), Subfamily(-virinae), Genus (-virus), Species (-virus). In order to localizeconserved regions in the fusion proteins one or a few candidates fromall viruses within an order are aligned first using an alignment toolsuch as the cobalt alignment tool(http://www.ncbi.nlm.nih.gov/tools/cobalt/). If stretches of conservedamino acids, such as ranging from 6 to 30 amino acids long, can beidentified these are considered as candidates for immunosuppressiveregions and are subjected to further investigation. If no candidates arefound in an order, the same procedure is applied to the family ofviruses. If still no candidates are found by testing different virusesbelonging to a family of viruses we move on to the subfamily of viruses.If we cannot localize regions of homology among the subfamily we thentest viruses from a genus and if we still cannot localize regions ofhomology we ultimately align as many possible individual viral sequencesfrom a single species of virus (up to 1400 individual viral sequences).In general regions of homology are identified by having at least 25%,more preferred at least 30%, preferably at least 40%, more preferred atleast 50%, more preferred at least 60%, preferably at least 70%, andeven more preferably at least 75% homology (i.e. sequence identity)within a given region.

Concerning step c., the dimerized peptide could be synthetic, themultimerized peptide could be displayed as dimerized or trimerizedfusion proteins either displayed alone or on membranes such as a viralparticle. Alternatively the multimerized peptides can be coupled to acarrier protein.

According to another aspect, the invention concerns a method fordecreasing or completely abrogating the immunosuppressive properties ofan immunosuppressive domain of a fusion protein of an enveloped RNAvirus containing a lipid membrane, said method comprising the steps of:

-   -   e. Mutating an immunosuppressive domain to produce at least one,        preferably a plurality of mutated peptides    -   f. Optionally dimerizing or multimerizing said at least one,        preferably plurality of mutated peptides;    -   g. Selecting at least one of said, preferably a plurality of        said mutated peptides by testing for reduced immunosuppressive        properties, preferably as shown by at least 25% reduction as        compared to a wildtype peptide mono-, di- or multimer        corresponding to the multimerization status of said mutated        peptides;    -   h. Mutating a fusion protein of an enveloped RNA virus        containing a lipid membrane to contain said selected mutated        peptide having reduced immunosuppressive properties;    -   i. Confirming expression by testing the whole viral envelope        protein encompassing said mutated fusion protein for capability        of being expressed by at least one of cellular or viral        surfaces.

According to an aspect, the invention concerns a method, furthercomprising the step of:

-   -   j. Using said viral envelope protein encompassing said mutated        fusion protein with reduced immunosuppressive properties as an        antigen for generation of an enhanced immune response.

A number of strategies are proposed for knock-out (i.e. decreasing orcompletely abrogating) of the immunosuppressive domain, these strategiescomprise, but are not limited to, mutating or modifying theimmunosuppressive domain into having the sequence of a mutant. Aknock-out may be achieved e.g. by mutation, deletion or insertion in animmunosuppressive domain. A mutation may be at least one exchange of anamino acid with another amino acid, at least one insertion, at least onedeletion, or a combination of one or more of these.

Mutants decreasing or completely abrogating the immunosuppressiveproperties will be identified by performing a complete or partlyscanning of said immunosuppressive peptide with either Isoleucine,Alanine Leucine, Asparagine, Lysine, Aspartic acid, Methionine,Cysteine, Phenylalanine, Glutamic acid, Threonine, Glutamine,Tryptophan, Glycine, Valine, Proline, Serine, Tyrosine, Arginine,Histidine, insertions, deletions or point mutations. Alternatively theliterature will be searched for mutations in said regions where saidmutation did not eliminate expression of the fusion protein on thesurface of the cell or viral envelope. Dimerized versions of saidmutants may be tested in a cell proliferation assay. The literatureprovides further details (as an example see Cross K J, Wharton S A,Skehel J J, Wiley D C, Steinhauer D A. Studies on influenzahemagglutinin fusion peptide mutants generated by reverse genetics. EMBOJ. 2001 Aug. 15; 20(16):4432-42).

According to an aspect, the invention concerns a method for identifyingan immunosuppressive domain in the fusion protein of an enveloped RNAvirus having a lipid membrane, said method comprising:

-   -   a. Identifying at least one well conserved domain among the        group consisting of the membrane associated domains of the        fusion protein and the surface associated domains of the fusion        protein;    -   b. Providing at least one peptide with the sequence of said        identified at least one well conserved domain;    -   c. Optionally dimerizing or multimerizing said at least one        peptide; and    -   d. Confirming the immunosuppressive activity of said at least        one optionally dimerized or multimerized peptide by testing said        at least one optionally dimerized or multimerized peptide for        immunosuppressive activity.

According to another aspect, the invention concerns an immunosuppressivedomain identified according to the invention.

According to another aspect, the invention concerns an immunosuppressivedomain selected among the sequences of Table 1 and Seq. Id. 1-200.

According to an aspect, the invention concerns a method for decreasingor completely abrogating the immunosuppressive properties of animmunosuppressive domain of the fusion protein of an enveloped RNA virushaving a lipid membrane, said method comprising the steps of:

-   -   e. Mutating an immunosuppressive domain to provide at least one        mutated peptide;    -   f. Optionally dimerizing or multimerizing said at least one        mutated peptide;    -   g. Selecting one of said optionally dimerized or multimerized        mutated peptides showing reduced immunosuppressive properties;    -   h. Mutating the fusion protein of the enveloped RNA virus to        contain said selected mutated peptide having reduced        immunosuppressive properties;    -   i. Confirming expression by testing the viral envelope protein        encompassing said mutated fusion protein for capability of being        expressed by at least one of cellular or viral surfaces.

According to an aspect, the invention concerns a mutated peptideproviding reduced immunosuppressive properties, said mutated peptidehaving a sequence according to Table 1 or any of Seq. Id. 201-203 orobtainable as said selected mutated peptide of the method according tothe invention.

According to an aspect, the invention concerns a method for generatingan enhanced immune response further comprising the step of:

-   -   j. Using said viral envelope protein encompassing said mutated        fusion protein with reduced immunosuppressive properties as an        antigen for generation of an enhanced immune response.

According to an aspect, the invention concerns a method for making anenvelope protein having diminished immunosuppressive activity,comprising: Mutating or modifying an immunosuppressive domain,identifiable according to the invention, of an enveloped RNA virus witha lipid membrane surrounding the core, to include a peptide obtainableaccording to the invention.

According to an aspect, the invention concerns an envelope proteinobtainable according to the invention.

According to an aspect, the invention concerns a mutated envelopeprotein obtainable according to the invention.

According to an aspect, the invention concerns a viral fusion proteinfrom an enveloped RNA virus with reduced immunosuppressive properties,said fusion protein encompassing a mutated peptide, said mutated peptidedisplaying reduced immunosuppression, and said mutated peptide replacingan un-mutated wildtype peptide having a sequence of an ISU of Table 1 oris selected among Seq. Id. 1-200.

According to an aspect, the invention concerns an envelope proteincomprising a mutated peptide according to the invention, said mutatedfusion protein being displayed on the surface of cells wherein saidmutated fusion protein is expressed.

According to an aspect, the invention concerns an enveloped RNA virus,different from a viruses selected among the group consisting ofRetrovirus, Lentivirus and Filovirus, wherein an immunosuppressivedomain has been modified or mutated to decrease or completely abrogatethe immunosuppressive properties of an immunosuppressive domain of thefusion protein.

According to an aspect, the invention concerns a virus selected amongthe vira of Table 1, wherein an immunosuppressive domain has beenmodified or mutated to decrease or completely abrogate theimmunosuppressive properties of an immunosuppressive domain of thefusion protein.

According to an aspect, the invention concerns an antigen obtainable byselecting a part of a mutated envelope protein according to theinvention, said part comprising the mutated domain of said envelopeprotein.

According to an aspect, the invention concerns a nucleic acid sequence,preferably recombinant, encoding a mutated envelope protein, an envelopepolypeptide or an antigen according to the invention.

According to an aspect, the invention concerns an isolated eukaryoticexpression vector comprising a nucleic acid sequence according to theinvention.

According to an aspect, the invention concerns a method for producing anantibody, said method comprising the steps of: Administering an entityselected among a mutated envelope, an envelope polypeptide, an antigen,a nucleic acid sequence or a vector according to the invention to ahost, such as an animal; and obtaining the antibody from said host.

According to an aspect, the invention concerns an antibody obtainableaccording to the invention.

According to an aspect, the invention concerns neutralizing antibodiesobtained or identified by the use of at least one envelope proteinaccording to the invention.

According to an aspect, the invention concerns a method formanufacturing neutralizing antibodies comprising the use of at least oneprotein according to the invention.

According to an aspect, the invention concerns a method formanufacturing humanized neutralizing antibodies, comprising the use ofat least one sequence selected among the sequences of Table 1 andsequences 201 to 203.

According to an aspect, the invention concerns a vaccine comprising avirus according to the invention.

According to an aspect, the invention concerns a vaccine compositioncomprising an envelope protein according to the invention.

According to an aspect, the invention concerns a vaccine compositioncomprising an entity selected among the group consisting of a mutatedenvelope protein, an envelope polypeptide, an antigen, a nucleic acidsequence, a vector and an antibody according to the invention, and inaddition at least one excipient, carrier or diluent.

According to an aspect, the invention concerns a medical compositioncomprising antibodies raised using a virus according to the invention.

According to an aspect, the invention concerns a pharmaceuticalcomposition comprising a mutated peptide, an envelope protein, a mutatedenvelope protein, an antigen, a nucleic acid sequence, a vector, anantibody or a vaccine composition according to the invention, and atleast one pharmaceutically acceptable excipient, diluents or carrier.

According to an aspect, the invention concerns a use of a mutatedpeptide, an envelope protein, a mutated envelope protein, an antigen, anucleic acid sequence, a vector or an antibody according to theinvention, for a medical purpose, such as for the treatment,amelioration or prevention of a clinical condition, and/or such as forthe manufacture of a medicament for the treatment, amelioration orprevention of a clinical condition.

According to an aspect, the invention concerns a method of treating orameliorating the symptoms of an individual, or prophylactic treating anindividual, comprising administering an amount of mutated peptide, anenvelope protein, a mutated envelope protein, antigen, nucleic acidsequence, vector or vaccine composition according to the invention.

DETAILED DISCLOSURE

Table 1 provides a list of viruses and their immunosuppressivedomain(s). Asterix denotes extremely conserved sequence in theimmunosuppressive domain for a given class, group, family or species ofviruses. New immunosuppressive domains identified and tested in CTLL-2assay for a given class, group, family or species of viruses are listed.Both the columns with “Putative ISU as described in this application foridentification of immunosuppressive domains” and “Peptides from domainsfrom fusion proteins exhibiting immunosuppressive activity (ISU)” arecandidates for domains which are immunosuppressive. Note that all of theentries of the latter column, were originally identified by theinventors as a member of the former column. Due to the redundancy, theentries of the latter column were not included in the former column.

1: The inventors have identified immunosuppressive domains in the fusionproteins among enveloped RNA viruses with a type II fusion mechanism.Insofar immunosuppressive domains have not been previously described fortype II enveloped RNA viruses. The immunosuppressive domain has beenidentified at two positions in the fusion protein in two differentgroups of viruses A: Co-localizing with the fusion peptide exemplifiedby the identification of an common immunosuppressive domain in thefusion peptide of Flavivirus (Dengue virus, west Nile virus etc.) and B:in the hydrophobic alpha helix N-terminal of the transmembrane domain inthe fusion protein exemplified by the finding of an immunosuppressivedomain in said helixes of Flaviridae e.g. Hepatitis C virus, Dengue,West Nile virus etc, cf. Table 1.2: The inventors have identified immunosuppressive domains in the fusionprotein among enveloped RNA viruses with type I fusion mechanism(excluding lentivirus, retrovirus and filovirus). This new positionco-localizes with the fusion peptide of said fusion protein asdemonstrated by the identification of a common immunosuppressive domainin the fusion peptide of all Influenza A and B types, cf. Table 1.3: The inventors have identified potential immunosuppressive domainslocated at various positions of type I enveloped RNA viruses (excludinglentivirus, retrovirus and filovirus) and enveloped RNA viruses withneither Type I nor type II fusion mechanism, cf. Table 1.

TABLE 1 Family Genus Species (group) Species (Strain)Putative ISU as identified Peptides from domains knock-out (K.O.)Name of envelope IU group and using the criteria from fusion proteinsmutants of the attchment/fusion fusion type described in this exhibitingimmuno- protein application for immunosuppressive suppressiveidentification of activity (ISU) domain (ISU) immunosuppressive domainsFlavi- Flavi-virus Aroa virus Bussuquara virus NRGWNNGCGLFGKFDRGWGNGCGDFGKG Envelope protein Group 1 Type viridae guape virus************** prME Fusion II Fusion Naranjal virus GDAAWDFGSVGGVFNSLGKprotein E mechanism **o****o*****oo*o** Dengue 1 GGTAWDFGSIGGVFTSVKGDRGWGNGCGLFGKG *o***************** **************KGSSIGMKMFESTYRGAKRMAIL G Dengue 2 GDTAWDFGSLGGVFTSVKG DRGWGNGCGLFGKG****************o** ************** KGSSIFKMFEATARGARRMAILG Dengue 3KGSSIGQMFETTMRGAKRMAILG DRGWGNGCGLFGKG **************GDTAWDFGSVGGVLNSLGK ******************* Dengue 4 GETAWDFGSVGGLLTSLGKDRGWGNGCGLFGKG ************oo***** **************KGSSIGKMFEATARGARRMAILG Japanese Japanese LGDTAWDFGSIGGVFNSIGDRGWGNGCGLFGKG encephalitis encephalitis ***o***************************** virus group virus Koutango virus LGDTAWDFGSVGGIFTSLGDRGWGNGCGLFGKG ************** Murray Valley LGDTAWDFGSVGGVFNSIGDRGWGNGCGLFGKG encephalitis ************** virus St. LouisLGDTAWDFGSIGGVFNSIG DRGWGNGCGLFGKG encephalitis ********************************* virus Usutu virus LGDTAWDFGSVGGIFNSVG DRGWGNGCGLFGKG**********o******** ************** West Nile Virus LGDTAWDFGSVGGVFTSVGDRGWGNGCGLFGKG **********o******** ************** Kokobera virusKokobera virus IGDDAWDFGSVGGILNSVG DRGWGNGCGLFGKG group unclassifiedKokobera virus group Modoc virus Modoc virus VGSAFWNSDQRFSAINLMD groupDRGWGNGCALFGKG Cowbone Ridge virus Jutiapa virus Sal Vieja virusSan Perlita virus mosquito-borne Ilheus virus LGDTAWDFGSVGGIFNSIGDRGWGNGCGLGFKG viruses Sepik virus TGEHSWDFGSTGGFFASVG DRGWGNGCGLFGKGNtaya virus Bagaza virus LGDTAWDFGSVGGFFTSLG DRGWGNGCGLFGKG groupTembusu virus LGDTAWDFGSVGGVLTSIG DRGWGNGCGLFGKG Yokose virusIGDDAWDFGSTFFIFNTIG DRGWGNGCGLFGKG Rio Bravo Apoi virusSSAFWNSDEPFHFSNLISII DEGWGNGCGLFGKG virus group Entebbe batGDDAWDFGSTGGIFNTIGKA DRGWGNGCGLFGKG virus Rio Bravo virusSSAYWSSSEPFTSAGIMRIL DRGWGNGCGLFGKG Saboya virus DRGWGNGCALFGKGGSSSWDFSSAGGFFGSIGKA Seaborne tick- Meaban virus GDAAWDFGSVGGFMTSIGRAborne virus DRGWGNHCGLFGKG group Saumarez Reef GETAWDFGSAGGFFTSVGRGvirus DRGWGNHCGLFGKG Tyuleniy virus GEAAWDFGSAGGFFQSVGRG DRGWGNHCGLFGKGSpondweni Zika virus LGDTAWDFGSVGGVFNSLGK DRGWGNGCGLFGKG virus group*************oo**o** Kyasanur forest VGEHAWDFGSVGGMLSSVGK disease virus*************o****** DRGWGNHCGLFGKG Langat virus VLGEHAWDFGSVGGVMTSIGDRGWGNHCGLFGKG Louping ill IGEHAWDFGSAGGFFSSIG virus **********o***oo*o*DRGWGNHCGLFGKG Omsk hemorrh- LGEHAWDFGSTFFGLSSIG agic fever virusDRGWGNHCGLFGKG Powassan virus VGEHAWDFGSVGGILSSVG *************o*****DRGWGNHCGFFGKG ************** Royal farm virus DRGWGNHCGLFGKG Tick-borneIGEHAWDFGSAGGFLSSIG encephalitis IGEHAWDFGSTFFGLTSVG virusIGEHAWDFGSTGGFLASVG DRGWGNHCGLFGKG Yaounde virus LGDTAWDFGSIGGVFTSLGDRGWGNGCGLFGKG Yellow fever Banzi virus VGSSSWDFSSTSGFFSSVGDRGWGNGCGLFGKG virus group Bouboui virus VGRSSWDFSSAGGFFSSVGDRGWGNGCGLFGKG Edge Hill virus Ugansa S virus Wesselsbron virusYellow fever MGDTAWDFSSAGGFFTSVG DRGWGNGCGLFGKG virus***o*************** unclassified Batu Cave virus NRGWGTGCFKWGIGDRGWGNGCGLFGKG Flavivirus Cacipacore virus NRGWGTGCGEWGLGCalbertado virus Cell fusing agent virus Chaoyang virus Chimeric Tick-borne encephalitis virus/Dengue virus 4 Culex theileri flavivirusDonggang virus Duck hemorrh- agic ovaritis virus Flavivirus Aedes/MO-Ac/ITA/2009 Flavivirus Anopheles/PV- Am/ITA/2009 Flavivirus CbaAr4001Flavivirus FSME Flavvivirus Phlebotomine/ 76/Arrabida/2007 Gadgets Gullyvirus Greek goat encephalitis virus Jugra virus Kadam virus Kamiti Rivervirus Kenougou virus Montana myotis leukoencephal- itis virus Mosquitoflavivirus Ngoye virus Nounane virus Phlebotomus flavivirus Alg_F19Phlebotomus flavivirus Alg_F8 Quang Binh virus Russian Spring-Summer encepha- litis virus Sokoluk virus Spanish sheep encephalitisvirus T'Ho virus Tai forest virus B31 Tamana bat virus Tick-borneflavivirus Wang Thong virus Flavivirus sp. Aeses flavivirusNRGWGTGCFEWGLG HVAGRYSKHGMAGIGSVWEDLVR Culex flavivirus NRGWGTGCFKWGIGVDKYRRFGTAGVGG Hepaci Hepatitis C Hepatitis C GLIHLHQNIVDVQYLYG E1/E2virus virus virus genotype PALSTGLIHLHQNIVDVQ 1 a Hepatitis CGLIHLHRNIVDVQYLYG virus genotype PALSTGLIHLHRNIVDVQ 1b Hepatitis CGLIHLHQNIVDVQYMYG virus genotype 2 PALSTGLIHLHQNIVDVQ Hepatitis CPALSTGLIHLHQNIVDVQ GLIHLHQNIVDVQYLYG virus genotype 3 Hepatitis CPALSTGLIHLHQNIVDVQ GLIHLHQNIVDVQYLYG virus genotype 4 Hepatitis CGLIHLHQNIVDTQYLYG virus genotype 5 PALSTGLIHLHQNIVDTQ Hepatitis CPALSTGLIHLHQNIVDVQ GLIHLHQNIVDVQYLYG virus genotype 6 All HepatitisGLIHLHQNIVDVQYLYG C virus Pesti virus Border disease Border diseaseNTTLLNGSAFQLICPYGWVGRVEC E1/E2 virus virus- SYFQQYMLKGQYQYSFDLEBorder disease virus-X818 Border disease virus 1 Border disease virus 2Border disease virus 3 Border disease virus isolates Bovine viralBovine viral NTTLLNGPAFQMVCPLGWTFTVSC diarrhea virus diarrhea virusSYFQQYMLKGEYQYWFDLE 1 1-CP7 Bovine viral diarrhea virus 1-NADLBovine viral diarrhea virus 1-Osloss Bovine viral diarrhea virus 1-SD1Bovine viral diarrhea virus isolates and strains Bovine viraldiarrhea virus type 1a Bovine viral diarrhea virus type 1b Pestivirusisolate 97-360 Pestivirus isolate Hay 87/2210 Pestivirusstrain mousedeer Pestivirus type 1 isolates Bovine viral Bovine viralSLLNGPAFQMVCPQGWTGTIEC diarrhea virus diarrhea virusDRYGQQYMLKGKWQYWFDLD 2 (BVDV-2) 2 Pestivirus sp. strain 178003Pestivirus sp. strain 5250Giessen-3 Bovine viral diarrhea virus-2 isolate SCP Classical swine Classical swine TLLNGSAFYLVCPIGWTGVIECfever virus fever virus SYFQQYMKGEYQYWFDLD Hog cholera virus strainZoelen unclassified Bovine viral TLLNGPAFQLVCPYGWTFTIEC Pestivirusdiarrhea virus 3 DNYFQQYMLKGKYQYWFDLEATD Chamois TLLNGSAFQMVCPFGWTGQVECpestivirus 1 DSYGQQYMLKGEYQYWFDLDAKD Porcine pesti-TLLNGPAFQLVCPYGWTFTIECD virus isolate SYYQQYIIKSGYQYWFDLTAKD BungowannahUnclassi- Barkedji virus fied Canine hepaci- Flavi- virus AAK-2011viridae GB virus A Douroucouli hepatitis GB virus A GBV-A-like agentsGB virus D GBV-C/HGV group GB virus C Hepatitis GB virus C-like virusHepatitis GB virus B Lammi virus Marmoset hepatitis GB virus ANakiwogo virus Turkey meningo- encephalitis virus TogaviridaeAlpha-virus Aura virus GVYPFMWGGAYCFCDTENTQVS E2/E1 Barmah Forest**********o****o**o*o* virus APFGCEIYTNPIRAENCAVGSIP Middelburg*****o*ooo*o**oo*oo*oo* virus SDFGGIATVKYSASKSGKCAVH Ndumu viruso***oooooo*ooooo*o*oo* Salmon pancreas FSTANIHPEFRLQICTSYVTCKGDdisease virus *oooooooo*oooo*ooooo*ooo Getah virus CHPP Mayaro virus*o** Trocara virus EEEV complex WEEV complex Fort Morgan virusHighlands J virus Sindbis virus Western equine encephalo- myelitis virusWhataroa virus VEEV complex Cabassou virus Mucambo virus Pixuna virusVenezuelan GVYPFMWGGAYCFCD equine enceph- *************** alitis virusGDCHPPKDHIVTHPQYHAQ ************o**o*o* AVSKTAWTWLTS *********oo*SFV complex Bebaru virus GVYPFMWGGAYCFCDTWNTQVS O'nyong-nyong**********o****o**o*o* virus APFGCEIYTNPIRAENCAVGSIP Ross River virus*****o*ooo*o**oo*oo*oo* Semliki forest SDFGGIATVKYSASKSGKCAVH viruso***oooooo*ooooo*o*oo* Una virus FSTANIHPEFRLQICTSYVTCKGD*oooooooo*oooo*ooooo*ooo CHPP *o** Chikungunya GVYPFMWGGAYCFCD virus*************** VHCAAECHPPKDHIVNY oo*o*oo*ooooooooo PASHTTLGVQDISATAMSWVo****oo******o****** Rubivirus Rubella virus Rubella virusACTFWAVNAYSSGGYAQLASYFNPG (strain BRD1) ***o*o****o**oo****o**o**Rubella virus GSYYK (strain BRDII) ****o Rubella virusQYHPTACEVEPAFGHSDAACWGFPT (strain ***o*o*o*o****o********o* Cendehill)DT Rubella virus ** (strain M33) MSVFALASYVQHPHKTVRVKFHT Rubella virus***oo*****o**o**o****** (strain RN-UK86) ETRTVWQLSVAGVSC Rubella viruso*o*********oo* (strain THERIEN) NVTTEHPFCNMPHGQLEVQVPP Rubella viruso*o*o**oo*o*o****o*oo* (strain TO-336 DPGDLVEYIMNYTGNQQSRW vaccine)****o******o*o****** Rubella virus GSPNCHGPDWASPVCQRHSPDCS(strain TO-336) ****o***o************** Rubella virus RLVGATPERPRLRLV(vaccine strain o***o**o**o**** RA27/3) DADDPLLRTAPGP *oo**********GEVWVTPVIGSQARKCGL oo*o**o**o*****o** HIRAGPYGHATVEM oo***********oPEWIHAHTTSDPWHP o**oooo*o***o*o PGPLGLKFKTVRPVALPR ****o***o**o*oo***ALAPPRNVRVTGCYQCGTPAL oooo**o*o*o**o******* EGLAPGGGNCHLTVNGEDVG***o*****o**oo*o*oo* LLNTPPPYQVSCGG ******o*o*o***RASARVIDPAAQSFTGVVYGTHT **o***oo*o************* TAVSETRQTWAEWAAAHWWQLTLGo*******ooo*****o******* Bunya- Hanta-virus Amur virusNPPDCPGVGTGCTACGVYLD Gn(G2)/Gc(G1) viridae Bayou virus**o****o********o*** Black Creek RKVCIQLGTEQTCKTIDSNDC Canal virus*oo*o*o*o*oo**oo*o*** Cano Delgadito DTLLFLGPLEEGGMIFKQWCTTTC virusQFGDPGDIM Calabazo virus GSFRKKCSFATLPSCQYDGNTVSG Catacamas virus*o***o*o***o*o*ooo**oo** Choclo virus ATKDSFQSFNITEPH Dobrava-**o****o**oooo* Belgrade virus GSGVGFNLVCSVSLTEC El Moro Canyon******o*o*ooo**** virus KACDSAMCYGSSTANLVRGQNT Hantaan virus****o*o***ooooo*o**o** Isla Vista GKGGHSGSKFMCCHDKKCSATGLV virus********o*o***ooo*ooo**o Khabarovsk AAAPHL virus *oo*** Laguna NegraDDGAPQCGVHCWFKKSGEW virus ***o*o*ooo***oo**** Limestone Canyon virusMonongahela virus Muleshoe virus Muju virus New York virus Oran virusPlaya de Oro virus Prospect Hill virus Puumala virus Rio Mamore virusRio Segunda virus Saaremaa virus Seoul virus Sin Nombre virusSoochong virus Thailand virus Thottapalayam virus Topografov virusTula virus Ortho- Anopheles A KHDELCTGPCPVNINHQTGWLT bunya- virus*o*o***o**oooooooo*o*o virus Anopheles B WGCEEFGCLAVSDGCVFGSCQD virus**o*oo**o*ooo**oo***** Bakau virus GNGVPRFDYLCHLASRKEVIVRKC Batama virus*o*ooo*ooo*oooo*ooooo*o* Bwamba virus SCAGCINCFQNIHC Caraparu virus*o**ooooooooo* Kaeng Khoi virus Kairi virus Madrid virus Main Drainvirus Marituba virus Nyando virus Oriboca virus Oropouche virusSathuperi virus Shamonda virus Shuni virus Simbu virus Tacaiuma virusTete virus Turlock virus unclassified Orthobunyavirus Akabane Sabo virusvirus Tinaroo virus Yaba-7 virus Bunyamwera Batai virus virusBirao virus Bozo virus Cache Valley virus Fort Sherman virusGermiston virus Guaroa virus Iaco virus Ilesha virus Lokern virusMaguari virus Mboke virus Ngari virus Northway virus Playas virusPotosi virus Shokwe virus Tensaw virus Tlacotalpan virus Xingu virusCalifornia California Encephalitis encephalitis virus serogrouopvirus LEIV California encephalitis virus-BFS-283 Chatanga virusInkoo virus Jamestown Canyon virus Jamestown Canyon-like virusJerry Slough virus Keystone virus La Crosse virus Lumbo virusMelao virus Morro Bay virus San Angelo virus Serra do Navio virusSnowshore hare virus South River virus Tahyna virus Trivittatus virusCaraparu Apeu virus virus Bruconha virus Ossa virus Vinces virusManzanilla Buttonwillow virus virus Ingwavuma virus Mermet virusMarituba Gumbo Limbo virus virus Murutucu virus Nepuyo virusRestan virus Wyeomyia Anhembi virus virus BeAr328208 virus Macaua virusSororoca virus Taiassui virus Phlebovirus Bujaru virus CandiruvirusChilibre virus Frijoles virus Punta Tor□Salehabad virus SandflyfeverNaples virus Uukuniemi viruso virus Rift Valley KTVSSELSCREGQSYWTfever virus **oo**oo*o**o*o** GSFSPKCLSSRRC *******ooooooENKCFEQCGGWGCGCFNVNPSCLF **o*o**o*oo*oo***ooo***o VHT **oWGSVSLSLDAEGISGSNSFSF **ooo*o**o*o*o*o*oo** RQGFLGEIRCNSE *o*****o**oo*AHESCLRAPNLVSYKPMIDQLEC *oo**oo**oooo*o*oo*ooo* DPFVVFERGSLPQTR**ooo*oo*o***o* QAFSKGSVQADLTLMFD **ooo*ooo*oooooo* CDAAFLNLTGCYSCNAG*o*o*o*oo*****oo* STVVNPKSGSWN *o*o**oooooo FFDWFSGLMSWFGGPLK*o***oo*o**oooooo unclassified Anhanga virus Phlebovirus Arumowot virusChagres virus Corfou virus Gabek Forest virus Itaproanga virusPhlebovirus Adria/ALB1/2005 Phlebovirus Adria/ALB5/2005 Phlebovirus AH12Phlebovirus AH12/China/2010 Phlebovirus AH15/China/2010 PhlebovirusB105-05 Phlebovirus B151-04 Phlebovirus B43-02 Phlebovirus B68-03Phlebovirus B79/02 Phlebovirus Chios-A Phlebovirus Cyprus PhlebovirusHB29/China/2010 Phlebovirus HN13/China/2010 Phlebovirus HN6/China/2010Phlebovirus Hu/Xinyang1/ China/2010 Phlebovirus Hu/Xinyang2/ China/2010Phlebovirus IB13/04 Phlebovirus JS2007-01 Phlebovirus JS24Phlebovirus JS26 Phlebovirus JS3/China/2010 Phlebovirus JS4/China/2010Phlebovirus JS6 Phlebovirus JSD1 Phlebovirus LN2/China/2010 PhlebovirusLN3/China/2010 Phlebovirus sandflies/Gr29/ Spain/2004 Phlebovirussandflies/Gr36/ Spain/2004 sandflies/Gr44/ Spain/2004 Phlebovirussandflies/Gr49/ Spain/2004 Phlebovirus sandflies/Gr52/ Spain 2004Phlebovirus sandflies/Gr65/ Spain/2004 Phlebovirus sandflies/Gr98/Spain/2004 Phlebovirus SD24/China/2010 Phlebovirus SD4/CHina/2010Phlebovirus tick/XCQ-2011 Phlebovirus XLL/China/2009 Rio Grande virusSalobo virus Sandfly fever sicilian virus Sandfly Sicilian Turkey virusUtique virus Phlebovirus sp. Phlebovirus sp. Be An 24262 Phlebovirus sp.Be An 356637 Phlebovirus sp. Be An 416992 Phlebovirus sp. Be An 578412Phlebovirus sp. Be Ar 371637 Phlebovirus sp. Co Ar 170255Phlebovirus sp. Co Ar 171616 Phlebovirus sp. GML 902878 Phlebovirus sp.Pa Ar 2381 Phlebovirus sp. PAN 479603 Phlebovirus sp. PAN 483391Phlebovirus sp. VP-161A Phlebovirus sp. VP-334K Phlebovirus sp. VP-366GOrthomyxo- Influenza- Influenza A INFA H1 GLFGAIAGFIEGGWTGWTYNAELLINF F#2 DELTA6: HA (HA1/HA2) Group 2 viridae virus A virusVLLENERTLDNAELLVLLENERTL GLFGAAGFIENGWEG Type I DYHD InF A H1-3: fusionINFA H2 GLFGAIAGFIEGGWQGWTYNAELL NAELLVLLENERTLD mechanismVLMENERTLDNAELLVLMENERTL FHD DYHD INFA H3 FIGGAIAGFIENGWEGWSYNAELLGLFGAIAGFIENGWEG VALENQHTIDNAELLVALENQHTI DLTD INFA H4GLFGAIAGFIENGWQGWSYNAELL VALENQHTIDNAELLVALENQHTI DVTD INFA H5GLFGAIAGFIEGGWQGWTYNAELL VLMENERTLDNAELLVLMENERTL DFHD INFA H6GIFGAIAGFIEGGWTGGLFGAIAG FIEGGWTGWTYNAELLVLLENERT LDNAELLVLLENERTLDMHDINFA H7 WSYNAELLVAMENQHTIDWSYNAE GLFGAIAGFIENGWEG LLVAMENQHLAD INFA H8GLFGAIAGRIEGGWSGWAYNAELL VLLENQKTLDNAELLVLLENQKTL DEHD INFA H9GLFGAIAGFIEGGWPGGLFGAIAG GIEGGWSGWAYNAELLVLLENQKT LDNAELLVLLENQKTLDEHDINFA H10 WTYQAELLVAMENQHTIDQAELLV GLFGAIAGFIENGWEG AMENQHTIDMAD INFA H11GLFGAIAGFIEGGWPGWSYANQLL VLLENEKTLDNAQLLVLLENEKTL DLHD INFA H12GLFGAIAGFIEGGWPGWAYNAELL VLLENQKTLKNAELLVLLENQKTL DEHD INFA H13GLFGAIAGFIEGGWPGWSYNAKLL VLLENDKTLDNAKLLVLLENDKTL DMHD INFA H14GLFGAIAGFIENGWQGWSYNAELL VALENQHTIDNAELLVALENQHTI DVTD INFA H15WSYNAELLVAMENQHTIDNAELL GLFGAIAGFIENGWEG VAMENQHTIDLAD INFA H16GLFGAIGFIEGGWPGWSYNAKLL VLIENDRTLDNAKLLVLIENDRT LDLHD Influenza-Influenza B All strains GFFGAIAGFLEGGWEGISSQIEL virus B virusAVLLSNEGIINQIELAVLLSNEG IINSED Influenza- Influenza C virus C virusParamyxo- Paramyxo- Avulavirus Avian paramyxo- GAIALGVATAAAVTAGF0 (F2/F1) viridae virinae virus 2 Yucaipa oooo*o*oo*o*oo** virusAvian paramyxo- virus 3 Avian paramyxo- virus 3b Avian paramyxo- virus 4Avian paramyxo- virus 5 Avian paramyxo- virus 6 Avian paramyxo- virus 7Avian paramyxo- virus 8 Avian paramyxo- virus 9 Newcastle disease virusPigeon para- myxovirus 1 unclassified Avulavirus Avian paramyxo-virus 10_Avian paramyxovirus duck/Miyagi/ 885/05 Avian paramyxo-virus penguin/ Falkland Islands/324/2007 Goosramyxovirus HZ Goose para-myxovirus JS/1/97/Go Goose para- myxovirus SF02 Henipavirus Hendra virusHendra virus horse/Australia/ Hendra/1994 Nipah virus unclassifiedHenipavirus Bat paramyxo- virus Eid.hel/GH45/ 2008 MorbillivirusCanine distemper virus Cetacean morbillivirus_ Dolphin morbillivirus_Pilot whale morbillivirus Porpoise morbillivirus Measles virusPeste-des- petits-ruminants virus Phocine dis- temper virus Phocine dis-temper virus 1 Phocine dis- temper virus-2 Rinderpest virus RespirovirusBovine para- influenza virus 3 Porcine para- myxovirus strainFrost Procine paramyxovirus strain Texas Human parain- fluenza virus 1Human parain- fluenza virus 3 Simian Agent 10 Sendai virus unclassifiedRespirovirus Atlantic salmon respirovirus Guinea pig parainfluenzavirus TS-9 Pacific salmon paramyxovirus Trask River 1983 Swineparainfluenza virus 3 Tursiops truncatus parainfluenza virus 1Rubulavirus Human parainfluenza virus 2 Human parainfluenza virus 2(strain Greer) Human parainfluenza virus 2 (strain Toshiba) Humanparainfluenza virus 4 Human parainfluenza virus 4a Human parainfluenzavirus 4b Mapuera virus Mumps virus Parainfluenza virus 5 Porcinerubulavirus Simian virus 41 unclassified Rubulavirus Porcineparainfluenza virus Tuhoko virus 1 Tuhoko virus 2 Tuhoko virus 3unclassified Atlantic salmon Paramyxovirinae paramyxovirus Beilong virusCanine parainfluenza virus Chimeric human parainfluenza virus rPIV3-2Fer-de-lance virus J-virus Menangle virus Mossman virus Murayama virusOvine parainfluenza virus 3 Pacific salmon paramyxovirus ParamyxovirusGonoGER85 Recombinant PIV3/PIV1 virus Reptilian paramyxovirusSalem virus Salmo salar paramyxovirus Snake ATCC-VR-1408 paramyxovirusSnake ATCC-VR-1409 paramyxovirus Tioman virus Tupaia paramyxovirusPneumovirus Human Human FLGLILGLGAAVTAGVA Group 3 respiratoryrespiratory ***oo**o*o*ooo*o* Type I syncytial syncytial virusTNEAVVSLTNGMSVL fusion virus A **o*****o**o*** mechanism HumanVIRFQQLNKRLLE respiratory **o***o*o**** syncytial virus REFSSNAGLT(strain RSB1734) ****o***o* Human MLTDRELTSIVGGM respiratory***oo**o*oooo* syncytial virus YVIQLPLFGVMDTDCW (strain RSB5857)*oo***oo**o**o** Human CLARADNGWYCHNAGSLSYFP respiratory**ooo*o**o*o****o*o** syncytial virus DTLKSLTVPVTSRECN (strain RSB6190)**oo***o*ooooo** Human YDCKISTSKTYVSTAVLTTMG respiratory*o*o*o***ooo*oo*o*oo* syncytial virus VSCYGHNSCTVIN (strain RSB6256)*****ooo**oo* Human GIIRTLPDGCHYISNKGVDRVQVGN respiratory***o*ooo**o*o**o*o*o*o*** syncytial virus TVYYLSKEVGK (strain RSB642)*o**oo*oo** Human PLSFPDDKFDVAIRDVEHSINQTRT respiratory**o**o*o*ooo*oo*ooo***ooo syncytial virus FLKASDQLL (strain RSB6614)*ooo**o** Human KIMTSKTDISSSVITSIGAIVSCYG respiratoryo*o***ooo*oo*o*oo*oo***** syncytial virus A strain Long LinkOut Humanrespiratory syncytial virus A2 Human respiratory syncytial virus B Humanrespiratory syncytial virus (subgroup B/ strain 18537) Human respiratorysyncytial virus (subgroup B/ strain 8/60) Human respiratorysyncytial virus 9320 Human respiratory syncytial virus B1 Humanrespiratory syncytial virus S2 Human respiratory syncytial virusstrain RSS-2 unclassified Human respiratory syncytial virus BovineAll strains FLGLILGLGAAVTAGVA Group 3 respiratory ***oo**o*o*ooo*o*Type I syncytial CLARADNGWYCHNAGSLSYFP fusion virus**ooo*o**o*o****o*o** mechanism YVIQLPLFGVMDTDCW *oo***oo**o**o**Metapneumo Avian All strains virus metapneumo- virus Human All strainsmetapneumo- virus Corona- Corona- Alphacorona- Alphacorona-RSAIEDLLFDKVKLSDVG S (S1/S2) Group 3 viridae virinae virus virus 1**oo****oo**ooo*o* Type I Coronavirus VPFYLNVQYRINGLGVT fusion group 1bo**ooooo**o**o*** mechanism Human corona- VLSQNQKLIANAFNNALHAIQvirus 229E **oo***o*ooo*oo*ooo** Human corona- TNSALVKIQAVVNANAvirus NL63 *oo**o*o*o***oo* Miniopterus bat AEAQIRDLINGRLTALNAYVSQQLcoronavirus 1 *oo******o***oo*oo*oo*** Minoopterus batSAAQAMEKVNECVKSQSSRINFCGN coronavirus HKU8 o*oo*oo*oo***oo*oo*oo***oPorcine epidemic GNHIIS diarrhea virus *o*oo* Rhinolophus batAPYGLYFIHFNYVP coronavirus HKU2 **o*oo*o*oo*o* Scotophilus batcoronavirus 512 unclassified Alphacorona- virus Betacorona-Betacoronavirus LQEAIKVLNHSYINLKDIGTYEYYV virus 1oo*oo*o**o*ooo*ooo*oo*o*o Coronavirus KWPWYVW group 2b *****o*Coronavirus group 2c Human corona- virus HKU1 Murine coronavirusPipistrellus bat coronavirus HKU5 Rousettus bat coronavirus HKU9Severe acute respiratory syndrome-related coronavirus recombinantSARSr-CoV SARS corona- virus Tylonycteris bat coronavirus HKU4unclassified Betacoronavirus Gammacorona- Avian corona- virus virusBeluga Whale coronavirus SW1 unclassified Alpaca corona- coronavirusesvirus CA08-1/ 2008 Bat coronavirus Bird droppings coronavirus Bovinerespiratory coronavirus Chicken enteric coronavirus Coronavirus AnasCoronavirus oystercatcher/ p17/2006/GBR Coronavirus red knot/p60/2006/GBR Ferret enteric coronavirus 1202 Ferret systemic coronavirus MSU-SFerret systemic coronavirus WADL Guangxi coronaviridae Humancoronavirus NO Human enteric coronavirus strain 4408 Kenya batcoronavirus Mink coronavirus strain WD1133 Parrot coronavirus AV71/99Quail coronavirus Italy/Elvia/2005 Tai Forest coronavirus unidentifiedcoronavirus unidentified human coronavirus Arena- Arena-virus LCMV-LassaIppy virus NALINDQLIMKNHLRDIMGIPYC GpC (Gp1/Gp2) Group 3 viridaevirus (Old Lassa virus *o**o***o*o***o*o**o*** Type I World) complexLujo virus FTWTLSDSEGKDTPGGYCLT fusion Lymphocytic oo*ooo*oo*ooo***o**omechanism choriomenin- KCFGNTAIAKCNQKHDEEFCDMLRL gitis virus***o*ooo****oo*oo****ooo* Mobala virus FDFN Mopeia virus oo*oMLQKEYMERQGKTPLGLVDLFVFS *ooo*oo**oo**oo*o*oooo*o Tacaribe virusAmapari virus FTWTLSDSEGKDTPGGYCLT (New World) Chapare virusoo*ooo*oo*ooo***o**o complex Flexal virus KCFGNTAIAKCNQKHDEEFCDMLRLGuanarito virus ***o*ooo****oo*oo****ooo* Junin virus FDFN Latino virusooo* Machupo virus MLQKEYMERQGKTPLGLVDLFVFS Oliveros virus*ooo*oo**oo**oo*o*oooo*o Paraná virus Pichinde virus Pirital virus Sabiávirus Tacaribe virus Tamiami virus Whitewater Arroyo virus Hepadna-Genus Hepatitis B HBV genotype A FNPLGFFPSHQLDPLF L and M and S Group 3viridae Orthohepa- virus HBV genotype B o***o*o*o*o*o*o* Fusion dnavirusHBV genotype C ADWDKNPNKDPWP mechanism- HBV genotype D o*o*o*oo*ooooNeither HBV genotype E MESITSGFLGPLLVLQAVFF type I nor HBV genotype Foooooooo*ooooo**oooo type II HBV genotype G LLTRILTIPQSLDSWWTSLNFLGGAHBV genotype H oooooo*oooo*oooo***o*o*oo Hepatitis B CPPTCPGYRWMCvirus alpha1 oo*o*****o*o Hepatitis B LFILLLCLIFLLVLLDYQ virus LSH/*oo*ooo*oo*oo*oooo chimpanzee Hepatitis B virus strain cpz  Hepatitis Bvirus subtype adr Hepatitis B virus subtype adw Hepatitis Bvirus subtype adyw Hepatitis B virus subtype ayw Rhabdo- Dimarhabdo-Ephemerovirus Bovine emhemeral LDGYLCRKQKWEVTCTETWYFVTD Glycoprotein GGroup 3 viridae virus fever virus *o*oo****o*ooo*o*****o*o NeitherKYQIIEVIPTENEC type I nor o***o**o*oooo* Type IILKGEYIPPYYPPTNCVWNAIDTQE fusion oo*oo*******oo*o**oooo** mechanismIEDPVTMTLMDSKFTKPC ooo*oooooo**o*oo** LHCQIKSWECIPV o**oo*o****o*SHRNMMEALYLESPD *oo*oo*o*oo*o** LTFCGYNGILLDMGEWWSIY o****oo**oooo******ELEHEKCLGTLEKLQNGE *****o**o*oo*oo*o* LDLSYLSPSNPGKHYAY**o***o*oo**oo*** IRAVCYYHTFSMNLD o**o*o*oo*oooo* VesiculovirusCarajas virus EWKTTCDYRWYGPQYITHSI Chandipura virus o*o****o*****o*o*o*Cocal virus LGFPPQSCGWASVTT Isfahan virus o****oo**oooooo Maraba virusVQVTPHHVLVDEYTGEWVDSQFING Piry virus ooooo*o*oooo*o*o*o*oooooorecombinant KC Vesiculovirus oo Spring viraemia of carp virus Vesicularstomatitis Alagoas virus Vesicular stomatitis Indiana virus Vesicularstomatitis New Jersey virus Lyssavirus Aravan virus Australian batlyssavirus Duvenhage virus European bat lyssavirus 1 European batlyssavirus 2 Irkut virus Khujand virus Lagos bat virus Mokola virusWest Caucasian bat virus Rabies virus Rabies virusGFTCTGVVTEAETYTNFVGYVT AB21 *o****o**o*oo*oooo*** Rabies virusSLHNPYPDYRWLRTVKTT AB22 *ooooooooooo***o* Rabies virusESLVIISPSVADLDPYDRSLHS AVO1 *ooo***oooo*o**ooo Rabies virusCKLKLCGVLGLRLMDGT BNG4 *ooo****oooo*ooo* Rabies virus ILGPDGNVLIPEMQSSBNG5 o**o*ooo*******o Rabies virus QHMELLESSVIPLVHPL China/DRV*ooo**o*ooo**oo** Rabies virus China/MRV Rabies virus CVS-11Rabies virus ERA Rabies virus Eth2003 Rabies virus HEP-FLURYRabies virus India Rabies virus Nishigahara RCEH Rabies virusOntario fox Rabies virus Ontario skunk Rabies virus PM Rabies virusred fox/08RS- 1981/Udine/2008 Rabies virus SAD B19 Rabies virussilver-haired bat-associated SHBRV Rabies virus strain Pasteur vaccinRabies virus strain Street Rabies virus vnukovo-32 Thailand geno-type 1 dog lyssavirus unclassified Bokeloh bat Lyssavirus lyssavirusEuropean bat lyssavirus Lyssavirus Ozernoe Shimoni bat virus Novirhabdo-Hirame virus rhabdovirus Infectious hematopoietic necrosis virusSnakehead rhabdovirus Viral hemorrhagic septicemia virus unassignedBangoran virus Rhabdo- Bimbo virus viridae Biven Arm virusFlanders virus Garba virus Klamath virus Malpais Spring virusNasoule virus Ngaingan virus Ouango virus Sigma virus Tupaia virusWongabel virus

According to an embodiment, the invention concerns a method foridentifying an immunosuppressive domain in the fusion protein of anenveloped RNA virus having a lipid membrane, said method comprising:

-   -   a. Identifying at least one well conserved domain among the        group consisting of the membrane associated domains of the        fusion protein and the surface associated domains of the fusion        protein;    -   b. Providing at least one peptide with the sequence of said        identified at least one well conserved domain;    -   c. Optionally dimerizing or multimerizing said at least one        peptide; and    -   d. Confirming the immunosuppressive activity of said at least        one optionally dimerized or multimerized peptide by testing said        at least one optionally dimerized or multimerized peptide for        immunosuppressive activity.

The at least one well conserved domain may be identified among domains,which are membrane associated and domains, which are surface associated.Naturally, a domain which is both membrane and surface associated may bea well conserved domain.

The fusion protein may be identified by searching NCBI taxonomy(http://www.ncbi.nlm.nih.gov/Taxonomy/), and selecting proteins of theFamily, Subfamily, Genus or Species to be investigated, and subsequentlysearch these for fusion or the specific name of the fusion protein, e.g.as listed in Table 1.

The dimerized peptide could be synthetic, the multimerized peptide couldbe displayed as dimerized or trimerized fusion proteins either displayedalone or on membranes such as a viral particle.

One way of testing the immunosuppressive activity of the at least onedimerized or multimerized peptide is to test the immunosuppressiveactivity of the fusion protein in the absence and presence of the atleast one dimerized or multimerized peptide, and comparing the results.

According to other embodiments, the invention concerns the method,wherein the identification of said at least one well conserved domain isdone among the group consisting of the surface associated domains of thefusion protein in one or more of the different conformations of thefusion protein undergoing fusion.

According to an embodiment, the invention concerns a method, wherein theenveloped RNA virus is not selected among Retroviruses, Lentiviruses orFiloviruses. In particular, according to an embodiment, the inventionconcerns a method, wherein said at least one well conservedimmunosuppressive domain is not located in the linker between the twoheptad repeat structures just N-terminal of the transmembrane domain inthe fusion protein of either Retrovirus, Lentivirus or Filovirus. Moreparticularly, according to an embodiment, the invention concerns amethod, wherein said at least one well conserved domain does not includesome of the 22 amino acids located N-terminal to the first of two wellconserved cysteine residues that are found in these structures in thefusion protein of either Retrovirus, Lentivirus or Filovirus. Thesecysteine residues are between 4 and 6 amino acid residues from oneanother and in many cases are believed to form disulfide bridges thatstabilize the fusion proteins.

According to other embodiments, the invention concerns the method,wherein said at least one well conserved domain is selected among thegroup consisting of Putative ISUs and Identified ISUs of Table 1 andSeq. Id. 1-200.

According to an embodiment, the invention concerns an immunosuppressivedomain identified according to the invention.

According to an embodiment, the invention concerns an immunosuppressivedomain selected among the sequences of Table 1 and Seq. Id. 1-200.

According to an embodiment, the invention concerns a method fordecreasing or completely abrogating the immunosuppressive properties ofan immunosuppressive domain of the fusion protein of an enveloped RNAvirus having a lipid membrane, said method comprising the steps of:

-   -   e. Mutating an immunosuppressive domain to provide at least one        mutated peptide;    -   f. Optionally dimerizing or multimerizing said at least one        mutated peptide;    -   g. Selecting one of said optionally dimerized or multimerized        mutated peptides showing reduced immunosuppressive properties;    -   h. Mutating the fusion protein of the enveloped RNA virus to        contain said selected mutated peptide having reduced        immunosuppressive properties;    -   i. Confirming expression by testing the viral envelope protein        encompassing said mutated fusion protein for capability of being        expressed by at least one of cellular or viral surfaces.

The envelope protein may be identified by searching NCBI taxonomy(http://www.ncbi.nlm.nih.gov/Taxonomy/) and selecting proteins of theFamily, Subfamily, Genus or Species to be investigated and subsequentlysearching these for envelope or the specific name for the envelopeprotein or the attachment and fusion protein, e.g. as listed in Table 1.

According to other embodiments, the invention concerns the method,wherein:

-   -   e. Said immunosuppressive domain is mutated to provide a        plurality of mutated peptides;    -   f. Said plurality of mutated peptides are optionally dimerized        or multimerized;    -   g. One of said optionally dimerized or multimerized mutated        peptides showing reduced immunosuppressive properties is        selected;    -   h. The fusion protein of the enveloped RNA virus is mutated to        contain said selected optionally dimerized or multimerized        peptide having reduced immunosuppressive properties;    -   i. Expression is confirmed by testing the viral envelope protein        encompassing said mutated fusion protein for capability of being        expressed by at least one of cellular or viral surfaces.

According to other embodiments, the invention concerns the method,wherein:

-   -   g. One of said optionally dimerized or multimerized mutated        peptide(s) is selected, which has reduced immunosuppressive        properties as shown by at least 25% reduction as compared to a        dimerized Wildtype peptide.

According to other embodiments, the invention concerns the method,wherein:

-   -   e. Said mutated immunosuppressive domain is mutated to provide a        knock-out mutant of Table 1 or selected among the sequences of        Seq. Id. 201-203.

According to an embodiment, a proven knock-out (i.e. a mutation of theimmunosuppressive domain abrogating the immunosuppressive properties ofthe peptide) from one family, genus, group and/or strain, may be usedfor another family, genus, group and/or strain.

According to an embodiment, the invention concerns a mutated peptideproviding reduced immunosuppressive properties, said mutated peptidehaving a sequence according to Table 1 or any of Seq. Id.-202 to 203 orobtainable as said selected mutated peptide of a method according to theinvention.

Preliminary experiments indicate the immunosuppressive domains may havea size of 4-30 amino acids.

According to an embodiment, the invention concerns a method forgenerating an enhanced immune response, comprising a method according tothe invention, and further comprising the step of:

-   -   j. Using said viral envelope protein encompassing said mutated        fusion protein with reduced immunosuppressive properties as an        antigen for generation of an enhanced immune response.

According to an embodiment, the invention concerns a method for makingan envelope protein having diminished immunosuppressive activity,comprising:

-   -   Mutating or modifying an immunosuppressive domain, identifiable        according to the invention, of an enveloped RNA virus with a        lipid membrane surrounding the core, to include a peptide        obtainable according to the invention.

The diminished immunosuppressive activity is suitably measured bycomparing to the immunosuppressive activity from an envelope of awildtype peptide. It is preferably demonstrated by an increasedproliferation of at least 25% in a cell proliferation assay ofhomodimers of said mutated peptide as compared to the homodimers of saidnon-mutated wildtype peptide at the same concentration. More preferablythe cell assay is either the CTLL-2 or the PBMC assay.

According to an embodiment, the invention concerns the method, formaking a envelope protein encompassing a mutated fusion protein from aenveloped RNA virus for medical use, such as therapeutic or prophylacticpurpose, preferably for use as a vaccine.

According to an embodiment, the invention concerns the method, formaking an enveloped protein encompassing a mutated fusion protein froman envelope RNA virus for vaccination purposes or for the generation ofneutralizing antibodies.

According to an embodiment, the invention concerns the method, whereinthe enveloped RNA virus has a fusion protein with a type II fusionmechanism.

According to an embodiment, the invention concerns the method, whereinthe enveloped RNA virus, preferably excluding lentivirus, retrovirus andfilovirus, has a fusion protein with a type I fusion mechanism and wherethe immunosuppressive domains co-localizes with the fusion peptide inthe fusion protein, preferably as demonstrated by the identification ofa common immunosuppressive domain in the fusion peptide of all H1 to H16of Influenza A and influenza B.

According to an embodiment, the invention concerns the method, whereinthe enveloped RNA virus, preferably excluding lentivirus, retrovirus andfilovirus, has a fusion protein with a type I fusion mechanism excludingviruses with a type I fusion mechanism where the ISU co-localizes withthe fusion peptide or the fusion protein has a structure that is neithera type I nor a type II fusion structure.

According to an embodiment, the invention concerns an envelope proteinobtainable according to the invention.

The immunosuppressive domain has so far been identified by the inventorsat two positions in two different groups of viruses A: Co-localizingwith the fusion peptide exemplified by the identification of an commonimmunosuppressive domain in the fusion peptide of all Flavivirus (Denguevirus, west Nile virus etc) and Influenza A and B viruses and B: in thehydrophobic alpha helix N-terminal of the transmembrane domain in thefusion protein exemplified by the finding of an immunosuppressive domainin said helixes of Flaviridae like e.g. Hepatitis C virus, Dengue, WestNile, Yellow fever.

The inventors have realized that the potential immunosuppressive domainsare located at various positions in the fusion protein identifiable by

-   -   1): The peptide is preferably located in the fusion protein of        enveloped RNA viruses;    -   2): The peptide is preferably capable of interacting with        membranes;    -   3): Preferably a high degree of homology in the primary        structure (sequence) of the peptide of said domain exists either        within the viral species itself, in the family of viruses or in        a group of viruses. This requirement is due to the        immunosuppressive domain being under a dual selection pressures,        one as an immunosuppressive entity ensuring protection of the        viral particle from the host immune system, another as a peptide        interacting with membranes; and/or    -   4): The position at the surface of the fusion protein at a given        conformation is preferably a feature of immunosuppressive        domains. This can be revealed either by position in a 3D        structure or by antibody staining of cells expressing the fusion        protein or on viral surfaces displaying the fusion protein.

According to an embodiment, the invention concerns a mutated envelopeprotein according to the invention.

According to an embodiment, the invention concerns a viral fusionprotein from an enveloped RNA virus with reduced immunosuppressiveproperties, said fusion protein encompassing a mutated peptide, saidmutated peptide displaying reduced immunosuppression, and said mutatedpeptide replacing an un-mutated wildtype peptide having a sequence of anISU of Table 1 or is selected among Seq. Id. 1-200.

According to an embodiment, the invention concerns the fusion protein,where the reduced immunosuppression is identified by comparing to theun-mutated wildtype peptide when said peptide is dimerized.

According to an embodiment, the invention concerns the fusion protein,wherein said immunosuppressive activity being determined by at least 25%reduction, more preferred at least 40% reduction, in proliferation ratein a cell proliferation assay using a homodimer of said un-mutatedpeptide compared to the monomeric version of said peptide at the sameconcentration.

According to an embodiment, the invention concerns the fusion protein,wherein said cell proliferation assay is selected among the groupconsisting of the CTLL-2 and the PBMC assay.

According to an embodiment, the invention concerns the fusion protein,wherein said fusion protein has a type I or type II fusion mechanism.

According to an embodiment, the invention concerns the fusion protein,wherein said fusion protein has neither a type I nor type II fusionmechanism.

According to an embodiment, the invention concerns the fusion protein,wherein said mutated peptide is located either in the fusion peptide orin a, preferably amphipatic, helix upstream of the C-terminaltransmembrane domain of said fusion protein.

The fusion peptide is a small membrane penetrating peptide located inthe fusion protein of enveloped viruses.

According to another embodiment, the invention concerns the viral fusionprotein, wherein said mutated peptide is derived from the fusion peptidefrom a flavivirus or Influenzavirus or from the amphipatic helix of theFlaviridae, such as the group consisting of Hepatitis C virus fusionprotein, Dengue virus fusion protein, and West Nile virus fusionprotein.

According to an embodiment, the invention concerns an envelope protein,said mutated fusion protein being displayed on the surface of cellswherein said mutated fusion protein is expressed.

According to an embodiment, the invention concerns the envelope protein,said mutated fusion protein being displayed on the surface of viral orviral like particles.

According to an embodiment, the invention concerns the envelope protein,having retained some fusogenic activity.

According to an embodiment, the invention concerns the envelope protein,wherein the fusogenic activity is measured by a technique for measuringcell-cell fusion, preferably selected among the group consisting ofcounting syncytial by light microscopy, resonance energy transfer basedassays, and indirect reporter gene using techniques or by measuringinfectious titers; alternatively, or in addition, the presence offusogenic activity may be indicated by the presence of at least one cellexpressing the modified envelope and one cell expressing the receptorand/or coreceptors being fused together.

According to an embodiment, the invention concerns an enveloped RNAvirus, different from a virus selected among the group consisting ofRetrovirus, Lentivirus and Filovirus, wherein an immunosuppressivedomain has been modified or mutated to decrease or completely abrogatethe immunosuppressive properties of an immunosuppressive domain of thefusion protein.

According to an embodiment, the invention concerns a virus selectedamong the vira of Table 1, wherein an immunosuppressive domain has beenmodified or mutated to decrease or completely abrogate theimmunosuppressive properties of an immunosuppressive domain of thefusion protein.

According to an embodiment, the invention concerns an antigen obtainableby selecting a part of a mutated envelope protein according to any ofthe preceding claims, said part comprising the mutated domain of saidenvelope protein.

According to an embodiment, the invention concerns an antigen comprisingan mutated immunosuppressive domain selected among the sequences ofTable 1 and Seq. Id. 201 to 202.

According to an embodiment, the invention concerns an antigen of theinvention furthermore harboring 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or9 or 10 or 11 point mutation(s) in any of the sequences of Table 1 or ofSeq. Id. 1-200.

According to an embodiment, the invention concerns an antigen, whichmediates fusion of virus to host cells.

According to an embodiment, the invention concerns an antigen, which isrecombinant or obtained by recombinant technology.

According to an embodiment, the invention concerns a nucleic acidsequence, preferably recombinant, encoding a mutated envelope protein,an envelope polypeptide or an antigen according to any of the precedingclaims.

According to an embodiment, the invention concerns an isolatedeukaryotic expression vector comprising a nucleic acid sequenceaccording to the invention.

According to another embodiment, the invention concerns the vector,which is a virus vector, preferably a virus selected among the groupconsisting of vaccinia virus, measles virus, retroviridae, lentivirus,baculovirus and adeno virus.

According to an embodiment, the invention concerns a method forproducing an antibody, said method comprising the steps of:

-   -   Administering an entity selected among a mutated envelope, an        envelope polypeptide, an antigen, a nucleic acid sequence or a        vector according to any of the preceding claims to a host, such        as an animal; and    -   Obtaining the antibody from said host.

According to an embodiment, the invention concerns an antibodyobtainable according to a method of the invention.

According to another embodiment, the invention concerns an antibody,which is specific for an entity selected among a mutated peptide, anenvelope protein, a mutated envelope protein, an antigen, a nucleic acidsequence or a vector according to any of the preceding claims.

According to an embodiment, the invention concerns neutralizingantibodies obtained or identified by the use of at least one envelopeprotein according to any of the preceding claims.

According to an embodiment, the invention concerns a method formanufacturing neutralizing antibodies comprising the use of at least oneprotein according to any of the preceding claims.

According to an embodiment, the invention concerns a method formanufacturing humanized neutralizing antibodies, comprising the use ofat least one sequence selected among the sequences of Table 1 andsequences 201 to 203

According to an embodiment, the invention concerns a vaccine comprisinga virus according to the invention.

According to an embodiment, the invention concerns a vaccine comprisingan envelope protein from a virus according to the invention.

According to an embodiment, the invention concerns a vaccine compositioncomprising an envelope protein according to any of the preceding claims.

According to an embodiment, the invention concerns a vaccine compositioncomprising a virus like particle (VLP).

According to an embodiment, the invention concerns the vaccinecomposition, wherein the virus like particle is produced ex vivo in acell culture.

According to an embodiment, the invention concerns the vaccinecomposition, wherein the virus like particle is partly or completelyassembled ex vivo.

According to an embodiment, the invention concerns the vaccinecomposition, wherein the virus like particle is generated in vivo in thepatient by infection, transfection and/or electroporation by expressionvectors.

According to an embodiment, the invention concerns the vaccinecomposition, comprising a vector derived from a measles or vacciniavirus.

According to an embodiment, the invention concerns the vaccinecomposition, comprising an expression vector for DNA vaccination.

According to an embodiment, the invention concerns the vaccinecomposition, comprising a purified envelope protein.

According to an embodiment, the invention concerns the vaccinecomposition, comprising a multimerized purified envelope protein.

According to an embodiment, the invention concerns the vaccinecomposition, comprising a dimerized purified envelope protein.

According to an embodiment, the invention concerns the vaccinecomposition, comprising a trimerized purified envelope protein.

According to an embodiment, the invention concerns a vaccine compositioncomprising an entity selected among the group consisting of a mutatedenvelope protein, an envelope polypeptide, an antigen, a nucleic acidsequence, a vector and an antibody according to any of the precedingclaims, and in addition at least one excipient, carrier or diluent.

According to an embodiment, the invention concerns the vaccinecomposition, further comprising at least one adjuvant.

According to an embodiment, the invention concerns a medical compositioncomprising antibodies raised using a virus according to the invention.

According to an embodiment, the invention concerns a pharmaceuticalcomposition comprising a mutated peptide, an envelope protein, a mutatedenvelope protein, an antigen, a nucleic acid sequence, a vector, anantibody or a vaccine composition according to any of the precedingclaims, and at least one pharmaceutically acceptable excipient, diluentsor carrier.

According to an embodiment, the invention concerns a use of a mutatedpeptide, an envelope protein, a mutated envelope protein, an antigen, anucleic acid sequence, a vector or an antibody according to any of thepreceding claims, for a medical purpose, such as for the treatment,amelioration or prevention of a clinical condition, such as for themanufacture of a medicament for the treatment, amelioration orprevention of a clinical condition.

According to an embodiment, the invention concerns a method of treatingor ameliorating the symptoms of an individual, or prophylactic treatingan individual, comprising administering an amount of mutated peptide, anenvelope protein, a mutated envelope protein, antigen, nucleic acidsequence, vector or vaccine composition according to any of thepreceding claims.

According to an embodiment, the invention may be used with human and/oranimal vira.

Table 2 below, provides the location of a number of identifiedimmunosuppressive domains.

TABLE 2 Localization of identified immunosuppressive domainsFamily (-viridae), Subfamily (-virinae), Genus (-virus) orSpecies (-virus) of Localization of prototype virusesimmunosuppressive domain Reference All Flavirus Protein ESeligman S J. Constancy and 98-DRGWGNXCGXFGKGXX-113diversity in the flavivirus fusion peptide. Virol J. 2008 Feb. 14; 5:27.All Flavirus Protein E FIG. 1 (e.g. Dengue 3)416-GDTAWDFGSVGGVLNSLGK-434 Schmidt A G, Yang P L, HarrisonS C. Peptide inhibitors of dengue-virus entry target a late-stage fusionintermediate. PLoS Pathog. 2010 Apr. 8; 6(4):e1000851. Hepatitis C E2Albecka A, Montserret R, Krey 71-GLIHLHQNIVDVQYLYG-87T, Tarr A W, Diesis E, Ball J K, Descamps V, Duverlie G, Rey F, Penin F,Dubuisson J. Identification of new functional regions inhepatitis C virus envelope glycoprotein E2.J Virol. 2011Feb.; 85(4):1777-92. Epub 2010 Dec. 8. Influenza A 1-16 HA2Cross K J, Wharton S A, Skehel Influenza B 1-GLFGAIAGFIENGWEG-16J J, Wiley D C, Steinhauer D A. Studies on influenzahaemagglutinin fusion peptide mutants generated by reversegenetics. EMBO J. 2001 Aug 15.; 20(16):4432-42.

According to an embodiment, an immunosuppressive domain may beidentified by its position, e.g. as indicated in Table 2.

According to an embodiment, the invention concerns an immunosuppressivedomain identified by its position.

According to an embodiment, the invention concerns an immunosuppressivedomain identified by its secondary, tertiary or quaternary structure inthe folded fusion protein.

According to an embodiment, the invention concerns an entity selectedamong the group consisting of a mutated peptide, an envelope protein, amutated envelope protein, an antigen, a nucleic acid sequence and avector, wherein an immunosuppressive domain identified by its position,has been modified or mutated in order to suppress its immunosuppressiveproperties.

All cited references are incorporated by reference.

The accompanying Figures and Examples are provided to explain ratherthan limit the present invention. It will be clear to the person skilledin the art that aspects, embodiments and claims of the present inventionmay be combined.

EXAMPLES Peptide Solutions

The peptides were either dissolved in water or in cases of low watersolubility, 5% DMSO solutions were used to dissolve the peptides.

Assay to Measure the Immunosuppressive Activity of Peptides Derived fromViral Surface Proteins or their Mutants

The peptides can be prepared by different means including, but notlimited to, solid phase synthesis commonly used for such purposes. Thepeptides can be dimerized using a cysteine residue either at the N- orC-terminal or in the middle of the peptide or by using any othermolecule or atom that is covalently bound to peptide molecules.

The peptides can be coupled to a carrier protein such as BSA by covalentbounds including, but not limited to, disulfide bridges between thepeptide cysteine residues and the carrier protein or through aminogroups including those in the side chain or Lysine residues.

The peptides can have non-viral derived amino acids added to theirC-terminal for increasing their water solubility.

Assay to Test the Immunosuppressive Activity of Peptides ExperimentDesign

Human Peripheral Blood Mononuclear Cells (PBMC) are prepared freshlyfrom healthy donors. These are stimulated by Con A (5 ug/mL) concomitantto peptide addition at different concentrations (i.e. 25 uM, 50 uM and100 uM). Cultures are maintained and lymphocyte proliferation ismeasured 72 hrs later by EdU incorporation and Click-iT labelling withOregon Green (Invitrogen, Denmark) as recommended by the manufacturer.The degree of activated lymphocytes is proportional to the fluorescencedetection.

CTLL-2 Assay

100.000 CTLL-2 cells are seeded pr. well in a 48 well-plate (Nunc) in200 uL of medium (RPMI+2 mM L-glutamine+1 mM Na-pyruvat+10% FCS+0.5ng/mL IL-2) 2 hours later the peptides are added to the wells. 24 hlater the cells are labeled using the Click-it reaction kit (Invitrogencat. # C35002). The fluorescence of the cells is measured on a flowcytometer. The degree of proliferation in each sample is proportional tothe detected fluorescence.

Test of Immunosuppression from Monomer and Dimeric Peptides

100.000 CTLL-2 cells were seeded pr. well in a 48 well-plate (Nunc) in200 uL of medium (RPMI+2 mM L-glutamine+1 mM Na-pyruvat+10% FCS+0.5ng/mL IL-2) 2 hours later the peptides were added to the wells. 24 hlater the cells were labeled using the Click-it reaction kit (Invitrogencat. # C35002). The fluorescence of the cells was measured on a flowcytometer. The degree of proliferation in each sample is proportional tothe detected fluorescence.

Quantification of Proliferation Inhibition

The degree of inhibition of proliferation of CTLL-2 cells is visualizedin the diagrams in the figures. The ratios are calculated by dividingthe number of labeled cells (growing cells) in cultures in presence ofpeptide with cultures in absence of peptides, but added the same volumeof the solute that was used to dissolve the peptides. That is in caseswhere the peptides were dissolved in 5% DMSO, the same volume of 5% DMSOwas added to the control cells.

FIGURES

FIG. 1 shows the result of an experiment using Influenza derivedpeptides, and the effect of the dimeric peptides on proliferation ofCTLL-2 cells. The peptides are coupled through an ss-bond involving thecysteine residues. The wt INF peptide inhibits synthesis of new DNA,whereas the non-IS #1 peptide has a much less and the non-IS #2 peptideno significant effect.

INF wt: GLFGAIAGFIENGWEGCGGEKEKEK

INF non-IS #1: GLFGAAGFIENGWEGCGGEKEKEK

INF non-IS #2: GLFAGFIENGWEGCGGEKEKEK

FIG. 2 shows the result of two independent experiments on Flavivirusderived peptides.

FLV IS/1 and FLV IS/2 are two independent experiments using thedimerized peptide: In both cases, a significant inhibition ofproliferation of CTLL-2 cells is evident, while the monomeric peptidehas no effect.

FLV IS/1 and FLV IS/2: dimeric DRGWGNGCGLFGKG

FLV IS mono/1: monomeric DRGWGNGCGLFGKG

Control peptide: a dimerized non-immune suppressive control peptide.

The concentrations are given in μM.

FIG. 3 shows another experimental result. The dimeric peptide derivedfrom Hepatitis C surface protein inhibits proliferation of T-cells in aconcentration dependent manner.

Hep C IS peptide has the sequence: PALSTGLIHLHQNIVDVQCGGEKEKEK

FIG. 4 shows yet an experimental result. The effect of the dimericpeptides derived from Flavi viruses on proliferation of CTLL-2 cells.The peptides are coupled through an ss-bond using the cysteine residues.FLV FUS non-IS is representative of a non-immune suppressive mutant.

Den H3: GDTAWDFGSIGGVFTSVGKCGGEKEKEK

FLV FUS non-IS: DRGWGNGCGDFGKG

APPENDIX

Classes of Enveloped RNA Viruses that Contain Human Pathogens

Flaviridae (Type II Fusion)

Flaviviridae have monopartite, linear, single-stranded RNA genomes ofpositive polarity, 9.6- to 12.3-kilobase in length. Virus particles areenveloped and spherical, about 40-60 nm in diameter.

Major diseases caused by the Flaviviridae family include:

-   -   Dengue fever    -   Japanese encephalitis    -   Kyasanur Forest disease    -   Murray Valley encephalitis    -   St. Louis encephalitis    -   Tick-borne encephalitis    -   West Nile encephalitis    -   Yellow fever    -   Hepatitis C Virus Infection

Existing Vaccines for Flaviridae

The successful yellow fever 17D vaccine, introduced in 1937, produceddramatic reductions in epidemic activity. Effective killed Japaneseencephalitis and Tick-borne encephalitis vaccines were introduced in themiddle of the 20th century. Unacceptable adverse events have promptedchange from a mouse-brain killed Japanese encephalitis vaccine to saferand more effective second generation Japanese encephalitis vaccines.These may come into wide use to effectively prevent this severe diseasein the huge populations of Asia—North, South and Southeast. The dengueviruses produce many millions of infections annually due to transmissionby a successful global mosquito vector. As mosquito control has failed,several dengue vaccines are in varying stages of development. Atetravalent chimeric vaccine that splices structural genes of the fourdengue viruses onto a 17D yellow fever backbone is in Phase III clinicaltesting.

Genus Flavivirus

Flaviviruses share a common size (40-65 nm), symmetry (enveloped,icosahedral nucleocapsid), nucleic acid (positive-sense, single strandedRNA approximately 10,000-11,000 bases), and appearance in the electronmicroscope.

These viruses are transmitted by the bite from an infected arthropod(mosquito or tick). Human infections with these viruses are typicallyincidental, as humans are unable to replicate the virus to high enoughtiters to reinfect arthropods and thus continue the virus life cycle.The exceptions to this are yellow fever and dengue viruses, which stillrequire mosquito vectors, but are well-enough adapted to humans as tonot necessarily depend upon animal hosts (although both continue haveimportant animal transmission routes as well).

Genus Hepacivirus (Type Species Hepatitis C Virus, the Single Member)

Hepatitis C is an infectious disease affecting the liver, caused by thehepatitis C virus (HCV). The infection is often asymptomatic, but onceestablished, chronic infection can progress to scarring of the liver(fibrosis), and advanced scarring (cirrhosis), which is generallyapparent after many years. In some cases, those with cirrhosis will goon to develop liver failure or other complications of cirrhosis,including liver cancer or life threatening esophageal varices andgastric varices. The hepatitis C virus is spread by blood-to-bloodcontact. Most people have few, if any symptoms after the initialinfection, yet the virus persists in the liver in about 85% of thoseinfected. Persistent infection can be treated with medication,peg-interferon and ribavirin being the standard-of-care therapy.Overall, 51% are cured. Those who develop cirrhosis or liver cancer mayrequire a liver transplant, and the virus universally recurs after thetransplant takes place. An estimated 180 million people worldwide areinfected with hepatitis C. Hepatitis C is not known to cause disease inother animals. No vaccine against hepatitis C is currently available.The existence of hepatitis C (originally “non-A non-B hepatitis”) waspostulated in the 1970s and proven in 1989. It is one of five knownhepatitis viruses: A, B, C, D, and E.

The hepatitis C virus is a small (50 nm in size), enveloped,single-stranded, positive sense RNA virus. There are six major genotypesof the hepatitis C virus, which are indicated numerically (e.g.,genotype 1, genotype 2, etc.). Based on the NS5 gene there are threemajor and eleven minor genotypes. The major genotypes diverged about300-400 years ago from the ancestor virus. The minor genotypes divergedabout 200 years ago from their major genotypes. All of the extantgenotypes appear to have evolved from genotype 1 subtype 1b.

The hepatitis C virus is transmitted by blood-to-blood contact. Indeveloped countries, it is estimated that 90% of persons with chronicHCV infection were infected through transfusion of unscreened blood orblood products or via injecting drug use or sexual exposure. Indeveloping countries, the primary sources of HCV infection areunsterilized injection equipment and infusion of inadequately screenedblood and blood products.

Genus Pestivirus Togaviridae Type II Fusion

The Togaviridae are a family of viruses, including the following genera:

Genus Alphavirus;

Alphaviruses have a positive sense single stranded RNA genome. There are27 alphaviruses, able to infect various vertebrates such as humans,rodents, fish, birds, and larger mammals such as horses as well asinvertebrates. Transmission between species and individuals occursmainly via mosquitoes making the alphaviruses a contributor to thecollection of Arboviruses—or Arthropod Borne Viruses. Alphavirusesparticles are enveloped, have a 70 nm diameter, tend to be spherical andhave a 40 nm isometric nucleocapsid.

There are two open reading frames (ORF's) in the genome, non-structuraland structural. The first is non structural and encodes proteins fortranscription and replication of viral RNA, and the second encodes threestructural proteins: the core nucleocapsid protein C, and the envelopeproteins P62 and E1 that associate as a heterodimer. The viralmembrane-anchored surface glycoproteins are responsible for receptorrecognition and entry into target cells through membrane fusion. Theproteolytic maturation of P62 into E2 and E3 causes a change in theviral surface. Together the E1, E2, and sometimes E3, glycoprotein“spikes” form an E1/E2 dimer or an E1/E2/E3 trimer, where E2 extendsfrom the centre to the vertices, E1 fills the space between thevertices, and E3, if present, is at the distal end of the spike. Uponexposure of the virus to the acidity of the endosome, E1 dissociatesfrom E2 to form an E1 homotrimer, which is necessary for the fusion stepto drive the cellular and viral membranes together. The alphaviralglycoprotein E1 is a class II viral fusion protein. The structure of theSemliki Forest virus revealed a structure that is similar to that offlaviviral glycoprotein E, with three structural domains in the sameprimary sequence arrangement. The E2 glycoprotein functions to interactwith the nucleocapsid through its cytoplasmic domain, while itsectodomain is responsible for binding a cellular receptor. Mostalphaviruses lose the peripheral protein E3, but in Semliki viruses itremains associated with the viral surface.

Genus Rubivirus; Genus Rubivirus Bunyaviridae Type II Fusion Mechanism

Bunyaviridae is a family of negative-stranded RNA viruses. Thoughgenerally found in arthropods or rodents, certain viruses in this familyoccasionally infect humans. Some of them also infect plants.

Bunyaviridae are vector-borne viruses. With the exception ofHantaviruses, transmission occurs via an arthropod vector (mosquitoes,tick, or sandfly). Hantaviruses are transmitted through contact withdeer mice feces. Incidence of infection is closely linked to vectoractivity, for example, mosquito-borne viruses are more common in thesummer.

Human infections with certain Bunyaviridae, such as Crimean-Congohemorrhagic fever virus, are associated with high levels of morbidityand mortality, consequently handling of these viruses must occur with aBiosafety level 4 laboratory. They are also the cause of severe feverwith thrombocytopenia syndrome.

Hanta virus or Hantavirus Hemorrhagic fever, common in Korea,Scandinavia, Russia, and the American southwest, is associated with highfever, lung edema and pulmonary failure. Mortality is around 55%.

The antibody reaction plays an important role in decreasing levels ofviremia.

Genus Hantavirus; Type Species: Hantaan Virus

Hantaviruses are negative sense RNA viruses in the Bunyaviridae family.Humans may be infected with hantaviruses through rodent bites, urine,saliva or contact with rodent waste products. Some hantaviruses causepotentially fatal diseases in humans, hemorrhagic fever with renalsyndrome (HFRS) and hantavirus pulmonary syndrome (HPS), but others havenot been associated with human disease. HPS cannot be transmittedperson-to-person. The name hantavirus is derived from the Hantan Riverarea in South Korea, which provided the founding member of the group:Hantaan virus (HTNV), isolated in the late 1970s by Ho-Wang Lee andcolleagues. HTNV is one of several hantaviruses that cause HFRS,formerly known as Korean hemorrhagic fever.

Genus Ortho-Bunya-Virus

The orthobunyaviruses are maintained in nature by sylvatic transmissioncycles between hematophagous mosquitoes and susceptible mammalian hosts,principally rodents and other small mammals. Several members of theCalifornia serogroup of orthobunyaviruses, including La Crosse (LAC) andTahyna (TAH) viruses, are significant human pathogens. LAC virus is animportant cause of pediatric encephalitis and aseptic meningitis in theMidwestern United States where approximately 100 cases are reportedannually; TAH virus, indigenous to central Europe, is associated withinfluenzalike febrile illnesses. La Crosse virus is a NIAID Category Bpriority pathogen.

The orthobunyaviruses are enveloped, negative-stranded RNA viruses witha tripartite genome comprised of large (L), medium (M), and small (S)segments The M segment encodes three proteins in a single open readingframe (ORF): two surface transmembrane glycoproteins, herein referred toas Gn (G2) and Gc (G1), respectively, to delineate their order in theprecursor polyprotein, and NSm, a protein of unknown function. Gn and Gcare thought to associate as a heteromultimer after cleavage of thepolyprotein.

Genus Phlebovirus; Type Species: Rift Valley Fever Virus

Phlebovirus is one of five genera of the family Bunyaviridae. ThePhlebovirus genus currently comprises over 70 antigenically distinctserotypes, only a few of which have been studied. The 68 known serotypesare divided into two groups: the Phlebotomus fever viruses (the sandflygroup, transmitted by Phlebotominae sandflies) comprises 55 members andthe Uukuniemi group (transmitted by ticks) comprises the remaining 13members.

Of these 68 serotypes, eight of them have been linked to disease inhumans. They are: Alenquer virus, Candiru virus, Chagres virus, Naplesvirus, Punta Toro virus, Rift Valley fever, Sicilian virus, and Toscanavirus. Recently identified is another human pathogenic serotype, theSFTS virus.

Rift Valley Fever (RVF) is a viral zoonosis (affects primarily domesticlivestock, but can be passed to humans) causing fever. It is spread bythe bite of infected mosquitoes, typically the Aedes or Culex genera.The disease is caused by the RVF virus, a member of the genusPhlebovirus (family Bunyaviridae). The disease was first reported amonglivestock in Kenya around 1915, but the virus was not isolated until1931. RVF outbreaks occur across sub-Saharan Africa, with outbreaksoccurring elsewhere infrequently (but sometimes severely—in Egypt in1977-78, several million people were infected and thousands died duringa violent epidemic. In Kenya in 1998, the virus claimed the lives ofover 400 Kenyans. In September 2000 an outbreak was confirmed in SaudiArabia and Yemen).

In humans the virus can cause several different syndromes. Usuallysufferers have either no symptoms or only a mild illness with fever,headache, myalgia and liver abnormalities. In a small percentage ofcases (<2%) the illness can progress to hemorrhagic fever syndrome,meningoencephalitis (inflammation of the brain), or affecting the eye.Patients who become ill usually experience fever, generalized weakness,back pain, dizziness, and weight loss at the onset of the illness.Typically, patients recover within 2-7 days after onset.

Approximately 1% of human sufferers die of the disease. Amongstlivestock the fatality level is significantly higher. In pregnantlivestock infected with RVF there is the abortion of virtually 100% offetuses. An epizootic (animal disease epidemic) of RVF is usually firstindicated by a wave of unexplained abortions.

Orthomyxoviridae Type I Fusion

The Orthomyxoviridae (orthos, Greek for “straight”; myxa, Greek for“mucus”)^(]) are a family of RNA viruses that includes five genera:Influenzavirus A, Influenzavirus B, Influenzavirus C, Isavirus andThogotovirus. A sixth has recently been described. The first threegenera contain viruses that cause influenza in vertebrates, includingbirds (see also avian influenza), humans, and other mammals. Isavirusesinfect salmon; thogotoviruses infect vertebrates and invertebrates, suchas mosquitoes and sea lice.

The three genera of Influenzavirus, which are identified by antigenicdifferences in their nucleoprotein and matrix protein infect vertebratesas follows:

-   -   Influenzavirus A infects humans, other mammals, and birds, and        causes all flu pandemics    -   Influenzavirus B infects humans and seals    -   Influenzavirus C infects humans and pigs

Paramyxoviridae Type I Fusion Mechanism

The fusion protein F projects from the envelope surface as a trimer, andmediates cell entry by inducing fusion between the viral envelope andthe cell membrane by class I fusion. One of the defining characteristicsof members of the paramyxoviridae family is the requirement for aneutral pH for fusogenic activity. A number of important human diseasesare caused by paramyxoviruses. These include mumps, measles, whichcaused 745,000 deaths in 2001 and respiratory syncytial virus (RSV)which is the major cause of bronchiolitis and pneumonia in infants andchildren. The parainfluenza viruses are the second most common causes ofrespiratory tract disease in infants and children. They can causepneumonia, bronchitis and croup in children and the elderly.

Human metapneumovirus, initially described in about 2001, is alsoimplicated in bronchitis, especially in children.

Genus Paramyxoviruses are also responsible for a range of diseases inother animal species, for example canine distemper virus (dogs), phocinedistemper virus (seals), cetacean morbillivirus (dolphins and porpoises)Newcastle disease virus (birds), and rinderpest virus (cattle). Someparamyxoviruses such as the henipaviruses are zoonotic pathogens,occurring naturally in an animal host, but also able to infect humans.

Hendra virus (HeV) and Nipah virus (NiV) in the genus Henipavirus haveemerged in humans and livestock in Australia and Southeast Asia. Bothviruses are contagious, highly virulent, and capable of infecting anumber of mammalian species and causing potentially fatal disease. Dueto the lack of a licensed vaccine or antiviral therapies, HeV and NiVare designated as biosafety level (BSL) 4 agents. The genomic structureof both viruses is that of a typical paramyxovirus.

Genus Pneumovirinae

-   -   Genus Pneumovirus (type species Human respiratory syncytial        virus, others include Bovine respiratory syncytial virus)    -   Human respiratory syncytial virus (RSV) is a virus that causes        respiratory tract infections. It is the major cause of lower        respiratory tract infections and hospital visits during infancy        and childhood. A prophylactic medication (not a vaccine) exists        for preterm birth (under 35 weeks gestation) infants and infants        with a congenital heart defect (CHD) or bronchopulmonary        dysplasia (BPD). Treatment is limited to supportive care,        including oxygen therapy.    -   In temperate climates there is an annual epidemic during the        winter months. In tropical climates, infection is most common        during the rainy season.    -   In the United States, 60% of infants are infected during their        first RSV season and nearly all children will have been infected        with the virus by 2-3 years of age.        http://en.wikipedia.org/wiki/Respiratory_syncytial_virus-cite_note-Glezen86-0        Of those infected with RSV, 2-3% will develop bronchiolitis,        necessitating hospitalization Natural infection with RSV induces        protective immunity which wanes over time—possibly more so than        other respiratory viral infections—and thus people can be        infected multiple times. Sometimes an infant can become        symptomatically infected more than once, even within a single        RSV season. Severe RSV infections have increasingly been found        among elderly patients.    -   RSV is a negative-sense, single-stranded RNA virus of the family        Paramyxoviridae, which includes common respiratory viruses such        as those causing measles and mumps. RSV is a member of the        paramyxovirus subfamily Pneumovirinae. Its name comes from the        fact that F proteins on the surface of the virus cause the cell        membranes on nearby cells to merge, forming syncytial.

Coronaviriridae Type I Fusion

Coronaviruses primarily infect the upper respiratory andgastrointestinal tract of mammals and birds. Four to five differentcurrently known strains of coronaviruses infect humans. The mostpublicized human coronavirus, SARS-CoV which causes SARS, has a uniquepathogenesis because it causes both upper and lower respiratory tractinfections and can also cause gastroenteritis. Coronaviruses arebelieved to cause a significant percentage of all common colds in humanadults. Coronaviruses cause colds in humans primarily in the winter andearly spring seasons. The significance and economic impact ofcoronaviruses as causative agents of the common cold are hard to assessbecause, unlike rhinoviruses (another common cold virus), humancoronaviruses are difficult to grow in the laboratory.

Coronaviruses also cause a range of diseases in farm animals anddomesticated pets, some of which can be serious and are a threat to thefarming industry. Economically significant coronaviruses of farm animalsinclude porcine coronavirus (transmissible gastroenteritis coronavirus,TGE) and bovine coronavirus, which both result in diarrhea in younganimals. Feline Coronavirus: 2 forms, Feline enteric coronavirus is apathogen of minor clinical significance, but spontaneous mutation ofthis virus can result in feline infectious peritonitis (FIP), a diseaseassociated with high mortality. There are two types of caninecoronavirus (CCoV), one that causes mild gastrointestinal disease andone that has been found to cause respiratory disease. Mouse hepatitisvirus (MHV) is a coronavirus that causes an epidemic murine illness withhigh mortality, especially among colonies of laboratory mice. Prior tothe discovery of SARS-CoV, MHV had been the best-studied coronavirusboth in vivo and in vitro as well as at the molecular level. Somestrains of MHV cause a progressive demyelinating encephalitis in micewhich has been used as a murine model for multiple sclerosis.Significant research efforts have been focused on elucidating the viralpathogenesis of these animal coronaviruses, especially by virologistsinterested in veterinary and zoonotic diseases.

SARS-Coronavirus

SARS is most closely related to group 2 coronaviruses, but it does notsegregate into any of the other three groups of coronaviruses. SARS wasdetermined to be an early split off from the group 2 coronaviruses basedon a set of conserved domains that it shares with group 2. A maindifference between group 2 coronovirus and SARS is the nsp3 replicasesubunit encoded by ORF1a. SARS does not have a papain-like proteinase 1.

Arenaviridae: Glycoprotein G2 is a Type I Fusion

Arenavirus is a genus of virus that infects rodents and occasionallyhumans. At least eight Arenaviruses are known to cause human disease.The diseases derived from Arenaviruses range in severity. Asepticmeningitis, a severe human disease that causes inflammation covering thebrain and spinal cord, can arise from the Lymphocytic choriomeningitisvirus (LCMV) infection. Hemorrhagic fever syndromes are derived frominfections such Guanarito virus (GTOV), Junin virus (JUNV), Lassa virus(LASV) causing Lassa fever, Machupo virus (MACV), Sabia virus (SABV), orWhitewater Arroyo virus (WWAV).^([1]) Arenaviruses are divided into twogroups; the Old World or New World. The differences between these groupsare distinguished geographically and genetically. Because of theepidemiological association with rodents, some arenaviruses andbunyaviruses are designated as Roboviruses.

-   -   LCMV-Lassa virus (Old World) complex:        -   Ippy virus        -   Lassa virus        -   Lujo virus        -   Lymphocytic choriomeningitis virus

LCMV infection manifests itself in a wide range of clinical symptoms,and may even be asymptomatic for immunocompetent individuals. Onsettypically occurs between one or two weeks after exposure to the virusand is followed by a biphasic febrile illness. During the initial orprodromal phase, which may last up to a week, common symptoms includefever, lack of appetite, headache, muscle aches, malaise, nausea, and/orvomiting. Less frequent symptoms include a sore throat and cough, aswell as joint, chest, and parotid pain. The onset of the second phaseoccurs several days after recovery, and consists of symptoms ofmeningitis or encephalitis. Pathological findings during the first stageconsist of leukopenia and thrombocytopenia. During the second phase,typical findings include elevated protein levels, increased leukocytecount, or a decrease in glucose levels of the cerebrospinal fluid).

Congenital Infection

Lymphocytic choriomeningitis is a particular concern in obstetrics, asvertical transmission is known to occur. For immunocompetent mothers,there is no significant threat, but the virus has damaging effects uponthe fetus. If infection occurs during the first trimester, LCMV resultsin an increased risk of spontaneous abortion. Later congenital infectionmay lead to malformations such as chorioretinitis, intracranialcalcifications, hydrocephalus, microcephaly or macrocephaly, mentalretardation, and seizures. Other findings include chorioretinal scars,optic atrophy, and cataracts. Mortality among infants is approximately30%. Among the survivors, two thirds have lasting neurologicabnormalities. If a woman has come into contact with a rodent duringpregnancy and LCM symptoms are manifested, a blood test is available todetermine previous or current infection. A history of infection does notpose a risk for future pregnancies.

Human-to-Human Transmission Through Organ Donation

In May 2005, four solid-organ transplant recipients contracted anillness that was later diagnosed as lymphocytic choriomeningitis. Allreceived organs from a common donor, and within a month oftransplantation, three of the four recipients had died as a result ofthe viral infection. Epidemiologic investigation traced the source to apet hamster that the organ donor had recently purchased from a RhodeIsland pet store. A similar case occurred in Massachusetts in 2008.Currently, there is not a LCMV infection test that is approved by theFood and Drug Administration for organ donor screening. The Morbidityand Mortality Weekly Report advises health-care providers to “considerLCMV infection in patients with aseptic meningitis and encephalitis andin organ transplant recipients with unexplained fever, hepatitis, ormultisystem organ failure.”

Hepadnaviridae: Fusion Mechanism Neither Type I Nor Type II

Hepadnaviruses are a family of viruses which can cause liver infectionsin humans and animals. There are two recognized genera

Hepadnaviruses have very small genomes of partially double-stranded,partially single stranded circular DNA. The genome consists of twouneven strands of DNA. One has a negative-sense orientation, and theother, shorter, strand has a positive-sense orientation.

As it is a group 7 virus, replication involves an RNA intermediate.Three main open reading frames are encoded (ORFS) and the virus has fourknown genes which encode the core protein, the virus polymerase, surfaceantigens (preS1, preS2, and S) and the X protein. The X protein isthought to be non-structural; however, its function and significance arepoorly understood.

Rhabdoviridae Fusion Mechanism Neither Type I Nor Type II

Rhabdoviruses carry their genetic material in the form of negative-sensesingle-stranded RNA. They typically carry genes for five proteins: largeprotein (L), glycoprotein (G), nucleoprotein (N), phosphoprotein (P),and matrix protein (M). Rhabdoviruses that infect vertebrates arebullet-shaped. The prototypical and best studied rhabdovirus isvesicular stomatitis virus.

Rhabdoviruses are important pathogens of animals and plants.Rhabdoviruses include RaV (Rabies virus), VSV (Vesicular stomatitisvirus). Rhabdoviruses are transmitted to hosts by arthropods, such asaphids, planthoppers, leafhoppers, black flies, sandflies, andmosquitoes.

ADDITIONAL REFERENCES

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1.-66. (canceled)
 67. A peptide providing immunosuppressive properties,said peptide comprising an amino acid sequence selected from thesequences shown in Table 1 or selected from SEQ ID NOS:1-200.
 68. Thepeptide according to claim 1 comprising the amino acid sequence of animmunosuppressive domain.
 69. A peptide having the amino acid sequenceof an immunosuppressive domain, said peptide identified by a method foridentifying an immunosuppressive domain in a fusion protein of anenveloped RNA virus having a lipid membrane, said method comprising: a)identifying at least one well-conserved domain among the groupconsisting of the membrane-associated domains of the fusion protein andthe surface-associated domains of the fusion protein; b) providing atleast one peptide with the sequence of said identified at least onewell-conserved domain; c) optionally dimerizing or multimerizing said atleast one peptide; and d) testing said at least one peptide or said atleast one optionally dimerized or multimerized peptide forimmunosuppressive activity.
 70. The peptide according to claim 69,wherein said at least one peptide is dimerized or multimerized.
 71. Thepeptide according to claim 3, wherein the identification of said atleast one well-conserved domain is performed using the group consistingof the surface-associated domains of the fusion protein in one or moredifferent conformations of the fusion protein undergoing fusion.
 72. Amutated peptide providing reduced immunosuppressive properties, saidmutated peptide comprising a mutated sequence selected from SEQ IDNOS:201-203 or a variant of an amino acid sequence selected from thesequences shown in Table 1 or selected from SEQ ID NOS:1-200, whereinsaid mutated sequence comprises from 1 to 4 amino acid substitutions, orat least one amino acid insertion, or at least one amino acid deletion.73. A mutated peptide providing reduced immunosuppressive properties,said mutated peptide being obtainable as a selected mutated peptide by amethod for decreasing or completely abrogating the immunosuppressiveproperties of an immunosuppressive domain of a fusion protein of anenveloped RNA virus having a lipid membrane, said method comprising thesteps of: a) mutating an immunosuppressive domain of said fusion proteinto provide at least one mutated peptide; b) optionally dimerizing ormultimerizing said at least one mutated peptide; c) selecting one ofsaid at least one mutated peptide or said at least one optionallydimerized or multimerized mutated peptide showing reducedimmunosuppressive properties; d) preparing a mutant of said at least onefusion protein or said at least one optionally dimerized or multimerizedmutated peptide containing said selected mutated peptide having reducedimmunosuppressive properties; and e) confirming expression of said atleast one mutated fusion protein or said at least one optionallydimerized or multimerized mutated peptide on a cell surface or a viralenvelope surface.
 74. The mutated peptide according to claim 73, whereina plurality of mutated peptides are provided in step (a), optionallyutilized in step (b), and utilized in steps (c)-(e).
 75. The mutatedpeptide according to claim 73, wherein said at least one mutated peptideor said at least one optionally dimerized or multimerized mutatedpeptide has immunosuppressive properties which are reduced by at least25% compared to a dimerized wildtype version of the same peptide(s). 76.The mutated peptide according to claim 73, wherein said mutatedimmunosuppressive domain is selected from SEQ ID NOS:201-203 or ismutated to provide a knock-out mutant of a sequence selected from thesequences shown in Table 1 or selected from SEQ ID NOS:1-200.
 77. Anenveloped RNA virus that is not a retrovirus, lentivirus, or filovirus,wherein an immunosuppressive domain of a fusion protein of the virus hasbeen modified or mutated to decrease or completely abrogateimmunosuppression by an immunosuppressive domain of the fusion protein.78. A virus selected from the viruses shown in Table 1 comprising animmunosuppressive domain that has been modified or mutated to decreaseor completely abrogate immunosuppression by an immunosuppressive domainof a fusion protein of the virus.
 79. A vaccine composition comprising avirus according to claim
 78. 80. A vaccine composition comprising anenvelope protein from a virus according to claim
 78. 81. A method oftreating or ameliorating a symptom caused by a virus infection of anindividual, or prophylactically treating an individual, the methodcomprising administering an effective amount of the vaccine compositionaccording to claim
 79. 82. A method of reducing or abolishing an immuneresponse in an individual, the method comprising administering to saidindividual an effective amount of the peptide according to claim
 67. 83.The method according to claim 82, wherein the immune response comprisescytokine secretion or proliferation of T-cells.